Devices and methods for purification, detection and use of biological cells

ABSTRACT

This invention relates to devices and methods for purifying, detecting and using biological cells. A variety of cell types including viable tumor, stem, immune and sperm cells can be purified from a complex biological sample using a column, including a pipette tip column. Methods of the invention can aid research, diagnosis and treatment of cancer. Purified viable cells can be detected on the column or eluted from the column and detected. Cells on a column can be used as a stationary phase for liquid chromatography. Cells may be removed, recovered and analyzed.

FIELD OF THE INVENTION

This invention relates to devices and methods for purifying, detectingand using biological cells. A variety of cell types can be purified froma complex biological sample using a column. The method can be performedquickly and viable cells can be recovered. Purified viable cells can bedetected on the column or eluted from the column and detected. Cells ona column can be used as a stationary phase for liquid chromatography.Cells may be removed, recovered, used and analyzed.

BACKGROUND OF THE INVENTION

The primary technology in use today for capturing and purifying cells ismagnetic beads. In this technology, a suspension of beads is used totreat a sample containing cells. The magnetic beads contain a tag orchemical entity that is selective for cells or for a certain cell typewithin the sample. After the cells become associated with the magneticbeads, a magnet is used to collect the magnetic beads and capturedcells. The magnetic beads may be re-suspended several times with washsolutions to clean the cells. Finally, a solution can be used to releasethe cells from the beads and a magnet separates the magnetic beads fromthe cells.

However, magnetic beads can negatively impact cell viability. Thisproblem is mitigated somewhat by the use of magnetic nanoparticles. Themagnetic-activated cell sorting (MACS) method available from MiltenylBiotec utilizes magnetic nanoparticles to isolate cells. Cellsexpressing particular surface antigens attach to the magneticnanoparticles.

For example, the isolation of circulating tumor cells (CTCs) from bloodis an area of very active interest at present. These cells which areshed into the vasculature from a primary tumor circulate in thebloodstream and constitute seeds for subsequent growth of additionaltumors (metastasis) in vital distant organs. CTCs present in thebloodstream of patients with cancer provide a potentially accessiblesource for detection, characterization, and monitoring ofnon-hematological cancers.

Magnetic bead methods are slow and do not always produce pure cellpopulations. In addition, cells isolated on magnetic beads are may notbe viable. There exists a need for a column technology that rapidlycaptures high concentrations of cells, particularly viable cells andthen recovers the cells at high purity for research, detection and forother uses.

In the instant invention, it was discovered that living cells can beused as a stationary phase for a new type of liquid chromatography. Inthis application, analyte reagents flow through a column in which cellsattached to the column medium serve as a stationary phase. Analytesinteract with the cell-based stationary phase. The extent of interactionof the analytes with the stationary phase can be measured and used.Later, the cell stationary phase may be recovered and analyzed. It isremarkable that in this type of chromatography, both mobile phaseanalytes and the stationary phase groups can be analyzed and measured.No previously-described chromatography systems have this capability.

Cells may vary in size and can be vulnerable to shear forces and stresswhich are exerted by a chromatographic device. These forces may bepresent while the cells are traveling through the chromatographic systemincluding the tubing, frits and column media. The forces may be presentwhile the cells are present on the column media.

Cells may be susceptible to contamination of various sorts includingchemical and biological contamination. However a chromatographic devicewith shielding for cells in the device is unknown. Chromatographicdevices cannot restrict or constrict the flow of liquid in any portionof the flow path including the inlet flow into the pump or column or theexit flow from the column. To restrict or constrict flow would make flowof liquid through the column impossible or at the very least,unpredictable, uncertain and uneven. Cells may be subjected toadditional shear forces and biological and mechanical stress when thechromatographic device is sealed. In addition to keep cells alive, theymust have nutrients, oxygen and removal of waste gas. Sealing achromatographic system may cause harm to cells, again in unpredictableways. There exists a need for a chromatographic system that preventscontamination of cells without harming the operation of thechromatographic system or harming the cells.

SUMMARY OF THE INVENTION

In the present invention, cells are purified from a biological sampleusing a column. The sample is passed through the column and cells arecaptured on the solid phase within the column. In some embodiments, thecolumn is within a sealed system that is not open to the ambientsurroundings. Sealing means that contaminants may not enter thechromatographic system. Contaminants cannot penetrate the device orenter the liquid phase. In some embodiments, gases may leave thechromatographic device by means of one directional check valves orsimilar one directional means. After construction of the sealed systemand after loading the buffers and sample into the column within thesystem, and during use of the device, the sample, buffers, fluid linesand reservoirs are not exposed. Contaminants from surroundings cannotpenetrate the device or enter the liquid phase. The sample is passedthrough the column and cells are captured on the solid phase within thecolumn. The solid phase can be a chromatography medium such as a gelresin or an impermeable resin. Following capture, the column is washedto remove material that is not specifically bound to the column medium.In some embodiments, cells can be recovered by passing an eluent throughthe column, while in other embodiments cells can be manipulated orinterrogated on the column.

Cells may be tagged for detection while attached to the column or may berecovered and subject to further manipulation, measurement or detection.In still other embodiments, cells pass through the column whiledesirable contaminants are captured.

Columns, methods and instruments of the invention may be used to captureand purify cells, clean reagents from solutions containing cells, ordetect or use cells. Practice of the invention facilitates the captureof living cells in a flow through column and keeping the cells in astable form, while the cells are flowing through the column and afterthe cells have been reversibly captured by the column. This is due tothe ability of being able to control the external chemical and physicalenvironment of the cells in a stable, non-disruptive manner. Once thecells are captured, flow through of reagents through the column can beused to allow interaction of reagents and cells to perform, optionalon-column manipulation of the cells. These manipulations includeinterrogation or measurement of interaction of reagents with the cellsor of the reagents that interact with the cells. The cells may be taggedwith a reagent. Tagging is the attachment of a chemical to the cell tomake the cell more detectable with different detectors. Optionally, thecells may be analyzed on the column through direct detection of thecells or materials within the cells. Or the cells may be destroyed andmaterials removed and analyzed. The cells may be recovered from thecolumn for further detection or interrogation.

In the methods of the invention, whole cells are isolated using a columnthat contains a bed of medium. In some embodiments, viable cells areisolated from the column. Cells are quite fragile and can rupture easilyfrom a variety of physical conditions such as encountering an object,shearing force, turbulence or incorrect solute concentration. Mechanicalcell lysis can be induced by a collision of the cells with micro beadsor with tubing and pumping devices associated with a sealed systemincluding peristatic pumps, positive or negative pressure pumps orgravity pumps. Even one breach of the cell membrane is enough to causecatastrophic damage to a cell. Viable cells can die in vitro simply fromincorrect storage, processing, transport, exposure to incorrecttemperature (heat or cold), pH, medium, vessel, collision with a sharpedge or small passage, etc. Cells may not have oxygen or nutrients tolive for a long time or may not be able to remove waste gases frombiological function leading to build up of conditions harmful to cellviability. Yet, in order to purify cells quickly with a column process,it is important that cells are passed through a column rapidly to beable to capture, wash and recover cells as quickly as possible. This isespecially true when the volume from which the cells are being capturedis large.

It is quite remarkable that intact cells and even viable cells can becaptured and purified using the columns and methods of the invention. Itis surprising that cells can remain intact even after subjecting them tothe methods of the invention. Specifically, cells purified via theinstant invention are subjected battering motion through a frittedcolumn containing a bed of medium, tubing and perhaps pumps, sometimesrepeatedly.

The advantages of invention over prior art include the following activemovement steps: 1. Active movement of cells to column bed functionalsite to capture live cells and to enable capturing cells in a rapidflowing stream: 2. Active movement of reagents to living cells whilereversibly attached to the column, to perform on-column chemicalreactions/interactions on living cells; to maintain cells in a livingstate, or to induce cells to perform biological activity and studybiological activity: 3. Active movement of washing fluids through thecolumn containing reversibly attached cells, to be able to rapidly andeffectively remove non-specific background cells and matrix moleculesfrom column: 4. Active movement of reagent through the column containingreversibly attached cells, to elute and recover living cells foranalysis or further use. All of the active movement steps are rapid withfast flowing fluids, but surprisingly do not harm the cells when thecolumns and methods of the invention are used. There exists a need forsuch a column technology that rapidly captures cells, particularlyviable or living cells, concentrates them and then recovers the cells athigh purity.

In still other embodiments, cells that are captured on the column may beused as a stationary phase. Reagents may be introduced into the columnin a mobile liquid phase. Reagents may interact or be retained by thecell stationary phase. These interactions may be measured. Analytereagents that interact with the stationary phase may be recovered andmeasured. Later, the cells may be removed from the column and analyzed.

The devices, columns, methods and instruments of the invention can beused with cells, including viable cells and cancer cells. In someembodiments, the column is a pipette tip column. In some embodiments,the column contains a solid medium. Cells can be manipulated orinterrogated while bound to the solid phase or cells can be eluted fromthe column. In some embodiments the flow of sample through the column isbidirectional. In some embodiments the flow rate is high so that thecell purification can be performed in 3 hours or less. In certainembodiments, cells can be isolated, purified, detected or used from asample in less than 30 minutes or even less. The methods of theinvention are quite versatile; many cell types can be isolated and awide variety of applications are possible.

Columns, method and instruments of the invention can be used in two waysor modes. In one mode the column the flow-through column may be used tocapture, tag, measure and recover living cells which may be furtherprocessed or analyzed. These cells may be recovered for R&D ordiagnostic and analytical purposes. In another mode, the column capturedliving cells may be used as a liquid chromatography column stationaryphase to measure and distinguish analyte reagent interactions with thestationary phase. After serving as a stationary phase, the cells may berecovered for R&D or diagnostic and analytical purposes. Cells ofvarious organ types may be contained on the column for study of theorgan and study of how chemicals interact with organ cells.

The devices and methods of the invention can be used for thepurification of cells, including viable cells, T cells, organ cells ofvarious types, stem cells and cancer cells and also including biologicalcells from organisms. A biological sample containing cells is passedthrough a column containing a solid phase and cells are captured on thesolid phase. In some embodiments, the column is a pipette tip column.Cells can be manipulated or interrogated while bound to the solid phaseor cells can be eluted from the column. In some embodiments the flow ofsample through the column is bidirectional. In some embodiments the flowrate is quite high so that the cell purification can be performed in 3hours or less. In certain embodiments, cells can be purified from asample in less than 30 minutes. The methods of the invention are quiteversatile; many cell types can be isolated and a wide variety ofapplications are possible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Stylistic depiction of flow path, nooks and traps in acolumn. FIG. 1A depicts an aspiration step. FIG. 1B depicts an expulsionstep.

FIG. 2. Elution of cells from the column shown in FIG. 1B.

FIGS. 3A-B Depiction of the column and method of the invention. FIG. 3Adepicts and aspiration step and FIG. 3B depicts and expulsion.

FIG. 4 is a depiction of an embodiment of a competition elutionstrategy.

FIG. 5 is a depiction of an embodiment of an antibody capture andrelease strategy.

FIG. 6 is a depiction of an embodiment of an aptamer capture and releasestrategy.

FIG. 7 is a depiction of one embodiment of a column having a cellstationary phase in a chromatographic system.

FIG. 8 is depiction of how cells are located on the surface of beadsthat are packed into a column to form cell stationary phase column.

FIG. 9 shows the features of a curve obtained from breakthroughchromatography.

FIG. 10 is a depiction of the different markers on the cell surface.These cell markers can interact with antibody analytes for cellpurification or forming a cell stationary phase. They can interact witha fluorescent tagged antibody. Cell markers may interact with analytes.

FIG. 11 is a depiction of a pipette tip chromatographic instrument witha pipette tip cell stationary phase column that can operate in abidirectional mode. The instrument can be used for cell purification anddiagnostic applications and cell stationary phase applications.

FIG. 12 is a depiction of a unidirectional flow column liquidchromatograph with a cell stationary phase column that operates usingunidirectional flow. This instrument also contains a bidirectional flowpump for loading the cells onto the stationary phase. The instrument canbe used for cell purification and diagnostic applications and cellstationary phase applications.

FIG. 13 is a graph showing the number of E. coli cells that are capturedon an agarose quaternary ammonium resin (Q) and a silica solid resin(A).

FIG. 14 depicts an embodiment of a closed column system for capture ofcells with a back and forth flowing system.

FIG. 15 depicts an embodiment of a closed column system for capture ofcells with a back and forth flowing system with two each feed andreceiving containers on each side of the column.

FIG. 16 depicts an embodiment of a closed chromatography column systemfor capture of cells with a back and forth flowing system with two eachfeed and receiving containers on each side of two optionally closedsystem columns.

FIG. 17 depicts an embodiment of a closed chromatography column systemfor capture of cells with a back and forth flowing system with two eachfeed and receiving containers on each side of two optionally closedsystem columns.

FIG. 18 depicts an alternative configuration of a sealed liquidchromatography column system for capturing cells using a flowing systemwith five each feed and receiving containers. The system may be usedwith unidirectional flow or back and forth flow. The pumping system maybe gravity, pressure, vacuum, or peristaltic pumping.

DETAILED DESCRIPTION OF THE INVENTION

In the methods of the invention, whole cells are isolated using a columnthat contains a bed of medium. In some embodiments, viable cells areisolated from the column. Cells are defined herein as membrane-boundstructures that occur as functional units of life (such as inunicellular organisms, e.g. bacteria, protozoa, etc.), or as structuralor fundamental units in a biological tissue specialized to perform aparticular function in multicellular organisms (e.g. plants andanimals). Self-replication is not a necessary property of cells asdefined herein; the definition includes entities such as viruses,parasites and exosomes.

Cells are quite fragile and can rupture easily from a variety ofphysical conditions such as encountering an object, shear force,turbulence or incorrect solute concentration, temperature and many otherconditions. Mechanical cell lysis can be induced by a collision of thecells with micro beads. In fact, this is a common method for cell lysis.However, even a little damage, even one breach of the cell membrane isenough to cause catastrophic damage to a cell. Viable cells can die invitro simply from incorrect storage, processing, transport, exposure toincorrect temperature (heat or cold), pH, medium, vessel, collision witha sharp edge or small passage, etc. Yet, in order to purify cellsquickly with a column process, it is important that cells are passedthrough a column rapidly to be able to capture, wash and recover cellsas quickly as possible. This is especially true when the volume fromwhich the cells are being captured is large.

Even though it is important to pass cells through a column quickly,prior art columns have not been able to do this (Braun, R., et al.,Journal of Immunological Methods 1982 54, 251-258, Bonnafous et al., J.Immunol. Methods 1983 Mar. 11; 58 (1-2):93-107 and Ohba, H., et al,Cancer Letters (2002) 184, 207-214). In a few cases, cells have beencaptured on a column using an incubation process where a small sample isapplied to the column and then the sample/column is held or incubated inorder to capture cells onto the column. Remarkably and in contrast tothe prior art, the instant method of passing cells through the columnrapidly without harm may also help or facilitate the improved capture ofthe cells from flowing samples and large samples.

It is remarkable that intact cells and even viable cells can be capturedand purified using the columns and methods of the invention. It issurprising that cells can remain intact even after subjecting them tothe methods of the invention. In certain embodiments, cells purified viathe instant invention are subjected to a repeated back and forth flowbattering motion through a fritted column containing a bed of medium.That is, cells can be passed rapidly through a column containing a bedof medium. Furthermore, it is surprising that cells can be manipulatedand reacted while captured on the column. The cells can remain attachedto the column while undergoing tagging or other reactions. The cells maybe used as a stationary phase for liquid chromatography. Analytereagents may interact with the attached cell stationary phase. Finally,the liquid chromatography stationary phase may be removed and recovered.It is remarkable the stationary phase cells may be recovered in aliving, viable state for further use or analysis.

The use of whole cells is an excellent format for cell-based assays fora number of reasons. First, it's possible to work with viable cells,which are closer to an in vivo environment than working with forexample, a single protein. In whole cells, targets such as cell surfaceproteins or protein complexes are likely to be intact and in theirnative state with respect to folding, etc. Interactions between cellscan be studied in some embodiments. Cell signaling pathways can betargeted.

In addition, using cells with column processes has a number ofadvantages. Columns can be operated in parallel and their operation canbe automated. Columns can be sterilized and operated in a sterileenvironment such as a laminar flow hood. Column processes are relativelygentle; there is no shaking, spinning or exposure to magnets. Thekinetics of drug-target interactions can be examined in a column asdescribed below. Cells, molecules or compounds can be added to columnsserially to examine the results of each addition. Cells can be isolatedquickly on the columns of the invention which aids in the retention ofviability.

Column processes with cells give an increased signal to noise ratio whencompared to other cell-based assays. Due to the large surface area ofthe beads, cells can be concentrated at the capture step to increasesignal. Wash steps remove non-specifically bound material, reducingnoise in a more consistent and complete manner. As a result, theseprocesses are more sensitive because of reduced noise and yield betterstatistical data because of the increased signal.

Another method to improve the sensitivity and signal to noise ratio isto improve the detectability of the cell. This is performed by reactingand attaching a detecting reagent to the cell while the cell is attachedto the column. By performing the tagging process in this manner, thereaction can be done more completely, which increases the sensitivity,and reproducibly, which reduces the noise, both of which increase thesignal to noise coming from the cells.

As described above, the columns of the invention contain a bed of mediumonto which the cells are captured. The bed can be comprised of beads orparticles held in the column by at least one frit below the bed. Incertain embodiments, the bed is retained in the column with two frits;one below the bed and one above the bed. It is quite surprising thatcells can pass through the frit(s) and the bed of medium and maintaintheir integrity and in some cases, even their viability.

Consider the physical environment of a liquid sample comprised of cellspassing through the frit and bed of medium within a column. The channelsthrough which a cell might flow are not open or linear. Instead, theflow path would consist of a variety of interwoven channels, each withvarying and perhaps restrictive diameters, and many possible dead endsmarked by repeated turns, bends, winding and twisting. This tortuouspath environment is advantageous for the capture of small moleculeanalytes because the fluid (containing the analyte) gets extensiveexposure to the column matrix. However, a cell travelling through thisenvironment could easily be trapped which can kill, damage and/orprevent the cell from being recovered. Adding one or two frits to thecolumn makes the flow path even more tortuous and restrictive. Ofcourse, physically trapping cells within the column matrix is anundesirable outcome quite distinct from targeted cell capture strategiessuch as affinity binding. Cells that become physically trapped cannot berecovered with an eluent or desorption solvent. Furthermore, if cellsare trapped, even temporarily, they could readily rupture or die. Thistrapping phenomenon was referred by Bonnafous et al. (supra) and teachesaway from successful purification of cells by the instant invention.

The columns of this invention have very low back pressures. The columnsare packed and constructed to produce these very low backpressures. Thecolumns of the invention have lower backpressures even compared tocolumns having low back pressure screen frits similar used in previouscolumn technology in which smaller column bed sizes and column bodysizes were used (U.S. Pat. No. 7,837,871. However, the backpressure ofthe columns of the invention is significantly lower than these earliercolumns. In addition, the columns are packed in such a way that the flowof cells through the column flow paths is less restricted and does notharm the cells.

Larger columns usually have higher backpressure than smaller columns.This problem is compounded when the columns are operated with lowpressure pumps. Pumps such as syringe pumps or pipette pumps applypositive pressure (head pressure) or vacuum to the column to force theflow of fluid through the column. The pressures applied are low andpumping fluids through large columns is limited. As a result, it is moredifficult to pump sample and buffers through large bed column resultingin slower flow rates and longer separation times. This can be problemfor capturing and recovering cells. Longer residence times in a columnwill harm the quality of the cells recovered and could prevent therecovery of cells, particularly viable cells.

FIGS. 1A and 1B illustrate the surprising nature of the invention. It isa stylistic depiction of the many potential hazards and pitfalls thatcould be encountered by cells travelling through a column usingbidirectional flow. FIG. 1A depicts an aspiration step in which the flowdirection 10 is upward. The matrix of the material (e.g., a polymer) isdepicted by closed squares 16. Cells cannot penetrate matrix 16. Theflow path through a column bed contains many potential nooks and trapsfor cells. A clear unrestricted flow path 12 enters and exits the bed.Some cells (e.g., 14) may be captured by the column in flow path 12. Asthe flow proceeds, many or most of the cells 18 enter dead end flowpaths 20 to trap the cells 22 in dead end or restricted passages 24.There are also nooks (e.g., 26) just off flow path 12 that may trap cell28.

FIG. 1B depicts the fate of cells resulting from back and forth flowthrough the column. The flow direction 30 is in a downward direction,reversed from upward direction 10 shown in FIG. 1A. Although increasedresidence time may allow a greater number of cells 32 to be captured,especially from a flowing stream, this reversal of the flow direction 34can also exacerbate the undesired trapping of many cells 36. It shouldbe noted that cell 28 remains trapped in nook 26.

FIG. 2 depicts the elution of cells from a column. The recovery of cellsfrom a column is attempted with a downward flow direction 38. Most ofthe cells 401 remain irreversibly trapped. A few cells 42 may berecovered but may or may not be intact. In addition to the risk of celltrapping, a person of skill in the art would expect the columnenvironment or materials to be inhospitable to cells. It is desirable torecover intact and even viable cells. Intact cells are defined herein ascells having no holes or ruptures in their membrane. The columnmaterials or surfaces, such as the frit or column walls might beincompatible with the cell integrity or viability. Protrusions presentin the column wall, bed or frit could easily damage or rupture cells.

FIGS. 3A and 3B depict a column and method of the invention. Because thecolumn is packed according to the methods of the invention and becausethe column is comprised of the frits described herein, it is not subjectto the pitfalls described above and shown in FIGS. 1 and 2. Cells in aliquid sample are passed through the column using back and forth flow.In these embodiments, the upper end of column 142 is operatively engagedwith pump 140 and sample 46 containing cells 44 is aspirated andexpelled through the lower end of the column. During the aspirationstep, the sample travels in direction 52, upwards in through lower frit56 into the bed of beads 48 and then continues through upper frit 150(FIG. 3A). During expulsion, the sample 46 travels back downward indirection 54, through upper frit 150, into the bed of medium, throughlower frit 56 and exits the bottom of column 142 (FIG. 3B). Theseaspirations and expulsions can be repeated multiple times, the desiredresult being that intact cells are captured by the medium.

However, columns of the invention capture cells in a reversible process.A vast majority of cells either flow through the column or arereversibly captured. Almost no cells are captured in restrictivechannels or dead end channels. The process of cell capture is reversibleand the cells are recoverable.

Almost no cells are damaged as they flow through the column, evenrepeatedly. Intuitively, it seems that the flow paths resulting fromback and forth flow would be even more perilous for cells thanunidirectional flow, especially when the goal is recovery of viablecells or even intact cells. Cells would pass through the column bed andfrit(s) multiple times from both directions, increasing the probabilityof cell damage or death.

This invention provides devices and methods for isolation of cells usinga column format. The cells can be eukaryotic or prokaryotic. The termcells, as used herein is not limited to self-replicating entities.Included in the definition are viruses, exosomes and parasites.

In certain embodiments, the isolated cells are viable. In someapplications, the maintenance of cell viability is less important. Forexample, cells purified on the column may be counted, labeled, analyzedby DNA sequencing, PCR or other assays.

The Sample

The starting sample is usually a heterogeneous mixture from which cellsare purified. The sample can be from any biological source and cancontain viable cells. For example, cells can be captured from biologicalfluids such as blood, urine, saliva, spinal fluid or semen, tissues suchas brain or tumor tissue and other samples such as fecal (stool) orhair. In certain embodiments, sample preparation steps are performedprior to the isolation of cells on a column. For example, when cells arecaptured from blood, the blood can be fractionated by centrifugation andonly the buffy coat loaded on the column. Alternatively, whole blood canbe diluted or loaded directly on the column. In certain embodiments, thedevices and methods can be used for the analysis of cells from crimescene samples.

In some samples, cells are free and exist individually in solution.There are other samples, such as tissues in which cells are aggregatedor form cell-cell adhesions. In addition there are cells that start offas tissues but then slough off to form free cells. Circulating tumorcells for example exist in blood and may form an adhesion to otherplaces in the body. Sample preparation techniques exist that canmechanically or chemically disrupt and dissociate cells in order to formsingle cell suspensions. These methods are gentle and in wide use. Kitsare available that use enzymatic digestion in combination withmechanical disruption and the option of heat. There are productsavailable from Miltenyi Biotec and Roche Life Sciences for example.

Cells isolated using methods and devices of the invention are notlimited to a particular cell type; cells captured by the methods of theinvention can be eukaryotic or prokaryotic cells. Eukaryotic cells canbe from protozoa, chromists, plants, fungi or animals such as mammals,amphibians, birds, fish, reptiles and invertebrates. Cells can beengineered or wild type.

A non-limiting list of cells that can be isolated by the columns of theinvention includes epithelial cells, hormone secreting cells, sensorytransducer cells, neuron cells, glial cells, lens cells, metaboliccells, storage cells, barrier function cells such as lung, gut, exocrineglands and urogenital tract, kidney cells, extracellular matrix cells,contractile cells, blood and immune system cells, germ cells, nursecells, interstitial cells, activated B-cells, mature B-cells, cytotoxicT-cells, helper T-cells, activated T-cells, natural killer (NK) cell,monocyte and macrophage, activated macrophage, endothelial cell, smoothmuscle cell, dendritic cell, mast cell, fibroblast (stromal), epithelialcell, adipocyte, stem cells, granulocytes, platelets, erythrocytescirculating tumor cells, Alexander cells, astroglia, B Lymphoblast, BLymphocyte, basophil, cortical neurons, cutaneous T cells, lymphocytes,embryonic cells, enterocytes, epithelial cells, transformed cells,immortalized cells, large T antigen, epithelial neuroendocrine,erythroblast, fetal, fibroblast, glial cell, glioblastoma, Hela cells,histocyte, human papillomavirus, hybridoma: e.g., helper T lymphocyte,keratinocyte, killer cell, large cell, lymphoblast, lymphoblast Blymphocyte, lymphoblast Human Immunodeficiency Virus, lymphocyte,medulloblastoma, megakaryoblast, melanocyte, melanoma, monoblast,myeloblast, neuroblast, neuroendocrine, osteoblast, pluripotent stemcell, pre-B lymphoblast, promyeloblast, retinoblastoma, Schwann cell,squamous cell, T lymphoblast, T lymphocyte, T-cell.

Cells isolated can be from any tissue. A non-limiting list of tissuetype examples follows. lung, ascites, bone marrow, bone, brain, cervix,colon, connective tissue, duodenum, eye, kidney: skin, kidney, liver,lung, lung: pleural effusion, mammary gland, ovary: ascites, ovary,pancreas: lymph node, pancreas, peripheral blood, pharynx, placenta,prostate, retinal pigmented epithelium, skin, spleen, stomach: derivedfrom metastatic pleural effusion, stomach, submaxillary salivary gland,testes, thyroid, tongue, urinary bladder, uterus, adrenal gland, airwayepithelium, aorta, bladder, blood, bone marrow, brain, breast, breastderived from metastatic site: pleural fluid, bronchiole, bronchus,carcinoma, cecum, cord blood, cornea, ectocervix, embryo, embryonickidney, endocervix, endometrium, epithelium, esophagus, eye, fetus,foreskin, gingival biopsy, heteromyeloma, intestine, kidney, lungadenocarcinoma, lymph node, lymph node derived from metastatic site:peritoneal effusion, mammary gland, marrow, mesencephalon, mesothelium,muscle, nasal septum, nervous, palatal, palatal mesenchyme, pancreas,peripheral blood, peritoneal effusion, peritoneum, peritonial effusion,pharynx: derived from metastatic site: pleural effusion, pleura,prostate, rectum, retina, retroperitoneal embryonal tumor,retroperitoneum, skin: derived from metastatic axillary node, skin:derived from metastasis on skin of thigh, small intestine, somatic cellhybrid, stomach, submaxillary, synovium, testis, thymus, thyroid,tonsil, trachea, trunk, umbilical vein, ureter, uterine, vagina,vascular, vein, vertebral epitheloid carcinoma and vulva.

Cells of a particular organ type or part of the body can be loaded ontoa column. These include cells from the heart, liver, kidney, bonemarrow, gut or from a spectrum of human tissues, including thecirculatory, endocrine, gastrointestinal, immune, integumentary,musculoskeletal, nervous, reproductive, respiratory, urinary systems andother types. The cells may be from a specific individual or from thegeneral population. Columns with these cells may be operated alone or inconcert with other columns containing cells from other organs orbiological systems. The columns can be operated in the chromatographicsystem in parallel or in series to mimic biological functions. Reagentscan be introduced into the columns to determine the interaction of thereagents and the effect on the cells or to study or use the cells asorgans.

The Columns

Columns used in the invention contain material capable of reversiblycapturing cells. Cells can be captured and eluted from the column. Insome embodiments, the eluted cells are viable.

The columns of the invention can be made in a wide range of sizes.Column bodies can range from a 10 μL pipette tip to a 200-mL column.These large volume columns are described in more detail below. Ofcourse, larger columns can be used to process larger liquid volumes. Forexample, a 20-ml pipette tip column containing 1 ml of resin canaccommodate approximately 19 ml of a biological liquid sample. Columnbed volumes can be in the range of 10 μL to 100 mL, 20 μL to 50 mL, 30μL to 10 mL, 40 μL to 5 mL, or 50 to 1 mL.

The columns can be comprised of beads or particles. In certainembodiments, the column medium can be a monolith, a filter or acombination of materials. In those embodiments which utilize beads, thebead size can be quite large, on the order of 100-900 microns or in somecases even up to a diameter of 3 mm. In other embodiments, the bead sizeis comparable to that used in conventional columns, on the order of45-150 microns. The average particle diameters of beads of the inventioncan be in the range of about 10 to 20 μm to several millimeters, e.g.,diameters in ranges having lower limits of 10 μm, 20 μm, 30 μm, 40 μm,50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 300 μm, or500 μm, and upper limits of 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80μm, 90 μm, 100 μm, 150 μm, 200 μm, 300 μm, 500 μm, 750 μm, 1 mm, 2 mm,or 3 mm.

Column formats and methods for use can vary significantly. In someembodiments, the columns are pipette tip columns. Pipette tip columnsare defined herein as columns capable of operative engagement with apipette, syringe, peristaltic, pressure, syringe pump or liquid handingrobot, or any pumping device that can impart positive and negativepressures to liquids or gases above the liquid to force the liquidthrough the column. Pipette tip columns have a lower end in whichliquids can be aspirated and expelled. In most embodiments, pipette tipcolumns have a frit to retain media located at the lower end and anoptional frit at the upper end. In certain embodiments, liquids arepassed through the column in a back and forth manner.

In other embodiments, the column can be in a cartridge or a column withend fittings. In this embodiment, of the column end fittings connect toliquid flowing tubes in and out of the column. If necessary, the ends ofthe columns contain frits to hold media in the column chamber. In otherembodiments, the columns have inlet and outlet fittings attached to theends. Tubing can be attached to these inlet and outlet fittings.

In other embodiments, the column is a non-compressed packed bed column.Non-compressed packed bed columns are used with unidirectional flow orthey can be used in bidirectional flow.

The columns may be used with any pumping device including the pumpslisted above and including pumping devices used in liquid chromatographinstruments including piston pumps and pressure type pumps.

In certain embodiments, the columns can be integrated into a multi-wellplate. In other embodiments, the column can be positioned within asyringe.

It was discovered that although bed compression was low with light forcepacked columns, the bed was still compressed and the beads werecompressed, restricting the flow of cells through the column. Weimproved capture of cells with affinity columns of the invention bypacking columns to form physical channels without constrictions or deadends. The bed can be positioned between two frits using a packing methodin which pressure is not used to compact the bed. Columns of theinvention do not have bed compression. With bed compression, beads aredeformed which causes them to fill the interstitial space. Column bedscan be compressed with a force to pack the column into the column space.This force can be applied with vacuum or pressure of liquid containingthe packing beads for physical compression of the beads into the columnchamber. With columns of the invention, the beads are not pressedtogether to form flow constrictions or dead-end flow spaces. The volumepacking density of the bed can be measured as a ratio of the volume ofbeads without having any direct contact causing the deformation of thebead divided by the volume of same amount of beads where the bed hasbeen compressed. As the volume of the column is decreased for the sameamount of beads, the volume packing density increases. A bed that hasbeen compressed 10% has a volume packing density of 1.00/0.90 whichequals 1.11. A bed that has been compressed 20% has a volume packingdensity of 1.00/0.80 which equals 1.25. A bed that has not beencompressed is 1.00/1.00 which equals 1.00. Columns of the invention thatcontain compressible beads have a volume packing density within therange of 1.00 to 1.05.

In certain embodiments, the columns are comprised of a more compressedpacked bed of medium. For example, a packed bed of medium might be usedfor enrichment columns in which cells pass through but contaminants arecaptured.

The space between the resin particles can be important. The space canincrease with a non-compressed packing of the column. This space mayprovide flow channels suitable for capture, washing and recovery ofcells without trapping the cells within the packing material. In manyembodiments, the columns can contain a mobile bed or a bed that packedin such a way that the beads are not compressed and the flow path willnot be restricted. Using this method, the resin can pack and formchannels in such a way that cells can move through the resin withreduced chance of damage and an increased chance of capture.

The column packing of the invention can be described functionally.Columns that are packed properly allow cells to pass through the bedwithout being trapped within the resin. In a column packed for use withcells (and lacking an affinity group for capture), at least 90% of thecells can pass through the column bed without being trapped. In someembodiments, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% of the cells can pass through the columnwithout being trapped. These numbers reflect the percentage of cellsthat can make it through the column in a single pass without beingtrapped.

In certain embodiments, the column medium is a hydrated gel resin. Gelresin is non-rigid and must be packed carefully. Also, in theseembodiments, the resin may be coated with a high boiling point liquidprior to use as described in U.S. Patent Application US20050045543.

It some embodiments, an impervious resin is used. The use of animpervious resin can be an improvement because cells are large and inmany cases, they cannot enter resin bead pores. Most prokaryotic cellsrange in size from 0.2 to 5.0 μm in diameter and most eukaryotic cellsrange in size from 1.0 to 100 μm in diameter. The reduction innon-usable surface area will decrease reagent costs as the capacity ofthe column is decreased. The use of a resin with the rigid structurewill also facilitate easier column packing procedures.

Example 18 describes the synthesis of a biotinylated silica resin.First, hydroxyl groups were added to the bead surface. Then, aminegroups were produced by reacting silanol with the hydroxyl groups. In athird step, biotin is reacted with the amine groups.

In most embodiments, cells are captured on the surface of the beads andnot in the interior of the beads however, in certain embodiments, thismay not be the case. Cells can be captured in large pore media. Thenumber of cells that can be captured depends on the diameter of shape ofthe cells and the available surface to which cells can be reversiblycaptured as a monolayer. Beads that are compressed not only restrict oreliminate the flow path through which cells can move without gettingtrapped. Compressed beads reduce the effective surface area of the beadwhich in turn, reduces the effective capacity of the resin to reversiblycapture cells.

The effective capacity of the column of the invention can be dependentupon accessible surface of the beads of the column. The effectivecapacity of a column should be distinguished from the actual capacity ofthe column, which is based on the total number of functional groups. Asthe beads become compressed and touch each other, some of the functionalgroups that are located on the surface of a bead may be inaccessible tocells flowing through the column and are not available for capture.Since columns of the invention contain beads that have limited or nocompression, the effective capacity of the column approaches thecapacity based on the total surface area of the media contained withinthe column. This is another improvement of columns of the invention overpreviously-described columns.

Column Frits

In certain embodiments of the invention, one or more frits are used tocontain the bed of medium within a column. The frits of the inventionare porous, since it is necessary for fluid to be able to pass throughthe frit. The pore size should be large enough to prevent plugging withcells or cell debris. It is important that the frit does not providedead-end or restricted-end flow paths that could potentially trap ordamage cells. It is desirable that the frit have little or no affinityfor liquids or cells with which it will come into contact during thecolumn use.

In certain embodiments, one frit (e.g., a lower, or bottom, frit)extends across the open channel of the column body. Often, the bottomfrit is attached at or near the open lower end of the column. A bed ofseparation medium is positioned inside the open channel and in contactwith the bottom frit. In many embodiments, a top frit is employed,however it is not mandatory. In certain embodiments, there is a gapbetween the bed of medium and the top frit. This gap is referred to as abed-frit gap.

Frits of various pores sizes and pore densities may be used provided thefree flow of liquid is possible and the solid phase is held in place.However, the frits must have specific porosity characteristics. It isnot only a matter of having sufficiently large pores. The pore shape isimportant as well. Pores cannot be destructive or restrictive to cells.

Frits of the invention preferably have pore openings or mesh openings ofa size in the range of about 5-500 μm. In certain embodiments, the poresize is in the range of 10-200 μm, 33-150 μm, e.g., about 33-43 μm. Fritpore sizes of 20, 33, 37 and 43 um pore size are acceptable. Of course,increasing the frit pore size can only be done if the packing materialretained.

The frits of the invention can be made from any material that has therequired physical properties as described herein. Examples of suitablematerials include polymers, fiber, fabric, plastic (including sinteredplastic), nylon, polyester, polyamide, polycarbonate, cellulose,polyethylene, nitrocellulose, cellulose acetate, polyvinylidinedifluoride, polytetrafluoroethylene (PTFE), polypropylene, polysulfone,PEEK, PVC, metal and glass. However, any suitable material that meetsthe above functional requirements can be used for the frit.

Certain embodiments of the invention employ a membrane screen as thefrit. The use of membrane screens can provide low resistance to flow andhence better flow rates, reduced back pressure and minimal distortion ofthe bed of medium. The membrane can be a woven or non-woven mesh offibers that may be a mesh weave, a random orientated mat of fibers i.e.a “polymer paper”, a spun bonded mesh, an etched or “pore drilled” paperor membrane such as nuclear track etched membrane or an electrolyticmesh.

Some embodiments of the invention employ a relatively thin frit. Thefrit or frits should be sufficiently thin such that cells will notbecome trapped or die within the frit during column operation. In mostembodiments, the frit thickness is less than 6000 μm or less than 4000μm (e.g., in the range of 20-4000 μm, 40-2000 μm, or 50-350 μm). Incertain embodiments, the frits are less than 200 urn thick (e.g., in therange of 20-200 μm, 40-200 μm, or 50-200 μm), or less than 100 urn inthickness (e.g., in the range of 20-100 μm, 40-100 μm, or 50-100 μm).However, thicker frits can also be used in some embodiments, frits up to1 mm, 2 mm, 3 mm, 4 mm, 5 mm and even 6 mm thick may be used if the poresize of the frit can be increased dramatically.

The frit can be attached to the column body by any means which resultsin a stable attachment. For example, the screen can be attached to thecolumn body through press fit, welding or gluing.

Frit and Column End Design for Cells for Different Types ofChromatography

Partitioning and frontal breakthrough chromatography are performed usingunidirectional flow while step gradient or displacement chromatographycan be done using either unidirectional or bidirectional flow.

Since flow is unidirectional for partitioning and frontal/breakthroughchromatography, there are specific design considerations necessary forthe frits and column ends. Specifically, the column end dead volume andflow distributor and the frit are critical to performing the separationsto prevent diffusion of the chromatographic analyte band and withouttrapping cells. In addition, in order to load the column, the column endflow diffusors and frits must accommodate back and forth flow withouttrapping the cells. Cells must be able to travel through the frits ineither direction without getting trapped. Conventional liquidchromatographic column ends and frits are not compatible with cellsbecause they will trap cells. Using a screen frit does not solve theproblem because this frit is flexible and the bed may move with thereverse direction flow. This will cause the column bed to move into thecolumn end flow distributor when the flow is reversed and could causethe cells to be damaged. The flow distributor must be deeper andcompatible with cells (not damage them) to prevent this even though thisdesign will result in band broadening.

The design described above can also be used for step gradient anddisplacement chromatography. In a liquid chromatographic format, theflow is likely to be unidirectional although the column flow could beused in unidirectional or bidirectional flow format and cell loading ofthe stationary phase is likely to be bidirectional. The dead volume ofthe column end and frit is not critical to performing the separationssince there is no chromatographic analyte band to diffuse. Nevertheless,the frit and column end should not trap cells.

Sterile Columns and System

In certain embodiments, the columns, reservoirs, tubing and flow pathsare sterile. In these embodiments, the columns materials can beassembled from sterile components in a sterile setting such as a cleanroom. Components can be sterilized by methods known in the art such asfiltration, irradiation, chemicals and heat.

Alternatively, terminal sterilization can be performed. Terminalsterilization is defined herein as sterilization of the manufacturedcolumns. In this embodiment, the columns and system can be sterilizedprior to use. Once the system with column is made sterile, the systemmay be sealed to maintain sterility. A sealed system does not letoutside materials enter the flow path of the system. There may beventing valve or other types of valves or membranes in the system butthese would only allow the out flow of materials and not allowcontamination of the system.

Column sterilization after manufacture can be performed by a number ofmethodologies. In one embodiment, columns can be assembled and thensterilized by autoclaving as described in the examples below.Alternatively, terminal sterilization treatment can be performed forexample, by irradiation.

The column hardware, media and system can be sterilized. For example,water swollen gels and other column media may be sterilized. Imperviousorganic and inorganic column materials may be sterilized. Substratesbased on silica and other inorganic materials may be sterilized.

Column Operation

In the methods of the invention, a sample containing cells is passedthrough a bed of medium or solid phase within a column. The cells arecaptured on the column medium while other sample constituents passthrough the column. The column with captured cells is washed and thepurified cells can be released from the column or manipulated on thecolumn.

Although it is not required, columns of the invention may be operatedusing back and forth flow. In some embodiments, liquids are aspiratedand expelled through the lower end of the column. This method isreferred to as back and forth flow or bidirectional flow. In theseembodiments, a pump, such as a liquid handling robot is operativelyengaged with the upper end of the column and liquids (such as thesample, wash and eluent) are aspirated and expelled through the lowerend of the column, such as a pipette tip column. Multiple aspirate expelsteps are often used with back and forth flow. Back and forth flow canalso be used in a packed bed column such as those used in a liquidchromatograph.

In other embodiments, unidirectional flow is used to pass liquidsthrough the column. In these embodiments, fluids are added to the upperend of the column and flow is in a downward direction through the columnand out the lower end. In some embodiments, the unidirectional flow isused to pass liquids from one end of the column to the other end. Insome embodiments, the inlet and outlet of the column may beinterchanged.

The sample can be passed through the column with the use of a pump, avacuum or even gravity. When unidirectional flow is used, liquids can bepassed through the column multiple times. That is, the flow-through canbe collected and loaded onto the column again.

The method can be performed in an automated or semi-automated fashion.In some embodiments, the method can be performed manually using ahand-held pipette or a syringe. The term “semi-automated” is defined asa process by which some steps are performed under electronic controlwhile other steps, such as moving the column from well to well areperformed manually. For example, a semi-automated method could beperformed using the electronic E4 pipette (Mettler-Toledo InternationalInc.) which is installed with firmware and software. The semi-automatedprocess can be performed in parallel with two or more columns.

The term “automated” is defined as a process by which sample processingis performed by a robotic system controlled by a computer program. Inthese embodiments, the timing for each processing step and programmingof the pumping device can be programmed such that the purification canbe performed in a walkaway fashion. This automation process may beperformed with columns in parallel. Even though backpressures are lowand the capture, wash and purification of cells is a difficult process,columns of the invention may be operated in parallel with automation.

Because of the very demanding design and performance requirements ofcapturing the cells in a reversible fashion, columns of the inventionmay have different flow properties. For example the backpressure of thecolumn may be very low. However, the backpressure may increase as thecolumn becomes loaded with cells that have been captured. Nevertheless,automated methods can be used successfully to purify and recover livecells with column, methods and apparatus of the invention.

Often, it is desirable to process a large volume of a liquid, e.g., abiological fluid. Sample volumes larger than the column bed or largerthan the column body can be processed by repeated aspiration andexpulsion of the sample. It is surprising that repeated aspiration andexpulsion can be performed without harming the sample. Alternatively,large sample volumes can be loaded onto the columns through the openupper end and collected from the open lower end. Sample loading can beperformed repeatedly as described above.

In some embodiments, the sample is comprised of a flowing stream. Inthese embodiments, the cells are captured by the column from a streamthat is pumped into the column and flows through the column. Because thecapture process is from a flowing stream, samples larger than the bedvolume of the column can be captured.

Columns of the invention are capable of capturing cells from largesample volumes, i.e. samples larger than one bed volume or one columnvolume. In some embodiments, the sample is comprised of a flowingstream. This is in contrast to previously-described columns whichrequire small volume samples limited to one bed volume and smaller(Braun et al., Bonnafous et al. and page Ohba et al. (supra)). Inaddition, Braun and Bonnafous teach that it is necessary to incubate thesample for several minutes before the separation process can begin. Itappears that their columns required incubation time for the cells tobecome captured by the resin and therefore were not capable of capturingcells from a flowing sample. Without being bound by theory, it appearsthe cells had to diffuse or undergo orientation to the affinity site inorder for the capture process to occur.

Intuitively, it seems that a slow flow rate would be advantageous forthe capture of cells on a column. There are several reasons for this.First, the cell surface protein must have the correct orientation to becaptured by the antibody (or other capturing group) on an affinityresin. Second, there must be sufficient time for the affinity group tobind and capture the cells. These two parameters improve as the flowrate is decreased. Furthermore, a slow flow rate would be gentler,lessening the chance of damage to the cells by the solid surface of themedium and/or column hardware.

However, even when slow flow rates are used, cells travel through thecolumn at relatively high linear velocities. A high linear velocitywould be expected to exacerbate the potential problems listed above. Forexample, a cell could become lodged in a dead end with greater force,making it more difficult to free the cell. While a cell travelling at arelatively slow velocity might slide or sidle around an obstacle, a celltravelling at a high velocity might be ruptured.

However, in the columns of the invention, rapid flow rates are possible.The use of rapid flow rates decreases separation time which positivelyimpacts cell viability. Although the cells may be subjected to morefrequent and harder collisions within the column body, they are able tosurvive even with repeated passes through the column and even with theuse of back and forth flow. Even through rapid flow rates would beexpected to decrease the opportunity for the cell capture, they allowcapture of the cells without damage.

Columns of the invention can accommodate a variety of flow rates, andthe invention provides methods employing a wide range of flow rates,oftentimes varying at different steps of the method. In variousembodiments, the flow rate of liquid passing through the media bed fallswithin a range having a lower limit of 0.1 mL/min, 0.5 mL/min, 1 mL/min,2 mL/min, or 4 mL/min and upper limit of 0.1 mL/min, 0.5 mL/min, 1mL/min, 2 mL/min, 4 mL/min, 6 mL/min, 10 mL/min, 20 mL/min, 30 mL/min,40 mL/min, 50 mL/min or greater.

A faster flow rate reduces the time for purification which isadvantageous for cell viability. On the other hand, a fast flow rate ismore likely to damage the cells.

It can be important to purify cells rapidly to maintain viability.Isolation of cells can be performed remarkably fast using the columnsand methods of the invention. Cells can be isolated in less than 3hours, less than 2½ hours, less than 2 hours, less than 90 minutes, lessthan 75 minutes, less than 60 minutes, less than 50 minutes, less than45 minutes, less than 40 minutes, less than 35 minutes, less than 30minutes, less than 30 minutes, less than 25 minutes, less than 20minutes, less than 15 minutes or less than 10 minutes. In otherembodiments, cell purification can take longer, particularly whenviability is not as important.

Columns of the invention have flow paths that allow the cells to becaptured from flowing streams. Capture is a fast process and so can beperformed with a flowing sample. This is a great improvement over thepreviously-described columns because capture from a flowing streamallows the capture of samples from volumes that are larger than the bedvolume and in some cases, larger than the column volume. In oneembodiment, the flowing sample stream is aspirated and expelled back andforth through the column at least once. In many embodiments, the sampleis passed back and forth through the column bed multiple times. There isno practical limit to the number of back and forth cycles althoughlengthy procedures may be harmful to the cells, particularly viablecells.

Cells can be captured from multiple sample aliquots processed in seriesor from multiple cycling from a large volume sample aliquot. Capturefrom a flow stream may be performed with unidirectional flow. In someembodiments, the capture is performed using slow flow rates, 100-200μL/min but the capture process is still successful with faster flowrates, up to 10 to 40 bed volumes/minute.

Cells traversing the column several times before being captured have agreater chance of becoming damaged or trapped. With each flow through ofthe sample through the column, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%,99.99% percent of the cells are either captured or travel through thecolumn and are not trapped or injured. With each cycle, additional cellsmay be trapped. For example if 99% of cells are unharmed with each passthrough the column, this number is reduced to 99% of 99% or 98% with thesecond pass (or one full back and forth cycle). With each additionalcycle, the reduction is another 2%.

When a sample containing cells is passed through the column, at least aportion of the cells are captured by the material within the column. Thesample may comprise a variety of cell types, e.g., blood and it may bedesirable to capture only one cell type. In some cases, rare cells suchas circulating tumor cells are captured on the column medium. In thesecases, the cells captured can be a very small percentage of the totalnumber of cells in the sample. In some embodiments, the number of cellscaptured can be relatively small.

In some embodiments, viable cells can be recovered from the column. Inthese embodiments, it is important to maintain the appropriateconditions for cell viability. Factors such as pH, buffering carbondioxide concentration, temperature and osmolarity must be considered inorder to keep cells alive and healthy. For example, viable cells can bestored in sterile buffers such as phosphate buffered saline (Ca/Mg⁺⁺free) or HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) orothers known in the art. These buffers can contain EDTA, HBSS (Hank'sbalanced salt solution), heat-inactivated fetal bovine serum and otherconstituents. One reference for such media can be found on the UCSF CellCulture Facility website.

However, viable cells are not required for all applications. Forinstance, it may be desirable to determine whether a particular celltype is present in a sample or to perform PCR on cells isolated usingthe columns and methods of the invention.

After the capture step, the columns can be washed with buffer or waterto remove any material that is not specifically bound to the columnmedium. The wash liquid can be passed through the column by any means orrate described above for the sample. The volume of the wash liquid canbe greater than that of the column. The wash step may be repeated onceor several times.

Following the column wash, the cells can be eluted from the column bypassing an eluent through the column. A variety of elution strategiesare described below. However, when viable cells are desired, the eluentand elution conditions must be chosen carefully to avoid or minimizeharm to the cells. The eluent can be passed through the column by anymeans described above for the sample. The elution step may be repeatedonce or several times. In certain embodiments, the eluent can beincubated on the column for a period of time to increase the efficiencyof cell elution. After the purified cells are eluted from the column,they can be analyzed by any means desired.

Because the columns are packed to minimize cell trapping, they can bevery efficient in isolating the desired cell type. It is possible tocapture at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95% and possible even 100% of the desiredcells from a particular sample.

After cells are isolated, an assay may be performed to determine cellviability or to count the number of viable cells.

In the columns and methods of the invention, cells can be captured fromrapidly flowing streams. As an example, for column with diametersranging from 2 mm-4 mm, capture is from cells moving through the columnat 0.05-20 mm/sec, 0.1-10 mm/sec, 0.2-5 mm/sec, 0.3-3 mm/sec, 0.4-2mm/sec and 0.5-1 mm/sec. The linear velocity at 0.1-10 mm/seccorresponds to absolute flow rates of about 100 μL/min to 10 mL/minrespectively. As an example, for column with diameters ranging from 4mm-4 mm, capture is from cells moving through the column at 0.05-20mm/sec, 0.1-10 mm/sec, 0.2-5 mm/sec, 0.3-3 mm/sec, 0.4-2 mm/sec and0.5-1 mm/sec. For some commonly used columns, the linear velocity at0.1-10 mm/sec corresponds to absolute flow rates of about 500 μL/min to5 mL/min respectively. As another example, for column with diametersranging from 10 mm-15 mm, capture is from cells moving through thecolumn at 0.05-20 mm/sec, 0.1-10 mm/sec, 0.2-5 mm/sec, 0.3-3 mm/sec,0.4-2 mm/sec and 0.5-1 mm/sec. For these columns, linear velocity at0.1-10 mm/sec corresponds to absolute flow rates of about 1 mL/min to100 mL/min respectively. As yet another example, for column withdiameters ranging from 15 mm-40 mm, capture is from cells moving throughthe column at 0.05-20 mm/sec, 0.1-10 mm/sec, 0.2-5 mm/sec, 0.3-3 mm/sec,0.4-2 mm/sec and 0.5-1 mm/sec. For these columns, linear velocity at0.1-10 mm/sec corresponds to absolute flow rates of 5 mL/min to 500mL/min respectively. Columns of the invention of other diameters operateat linear and absolute flow rates corresponding relative to the examplesgiven.

The following steps are example of a chromatographic procedure forpurification of cells for recovery and detection.

-   1. Treat or activate column or cells to make the column suitable to    be able to capture cells.-   2. Load cells onto column from a flowing stream.-   3. Wash nonspecific bound materials from the column using a flowing    stream.-   4. Optionally tag cells with a reagent that reacts with a group on    the surface of the cell. The tag reacts with an attachment group or    entity that is optionally different than the attachment group used    to capture the cell to the column.-   5. Optionally measure cells with the cells attached to the column.    Optional detectors are infrared, surface and transmission VIS/UV,    fluorescence, chemiluminescence spectrophotometers.-   6. Optionally lyse cells or elute the components of the cells for    analysis. The lysing may be done partially, or over a long time    period using gentle conditions to remove components of the cell for    processing and/or analysis.-   7. Remove and recover cells for analysis of the cells and/or further    R&D processing, growing or transforming of the cells or further R&D    and/or therapeutic use of the cells.    Active Movement

The movement of reagents and cells in cell-based assays and magneticbead assays is based on diffusion. Reagent and cells travel throughsolution by diffusion. This process is attractive because it is notdisruptive to cells i.e. the processes treat cells gently. This isimportant because rapid changes in the chemical and physical environmentof the cells can injure or kill the cells.

But with diffusion, fine control of the chemical environment can bedifficult to attain because the addition and removal of reagents is slowand residual materials cannot be removed easily. It may be difficult tochange reagents that are in contact with the cells or control changes inthe buffer concentration or pH. The diffusion process can be slow andcells can die simply through natural causes.

We use the term active movement to describe the use of pumps to forcecells, by a fluid pumping action, to travel in a flowing stream to andfrom tubing and column connections, fittings, frits and column media.Active movement can be performed by unidirectional flow andbi-directional flow. The term active movement is also used to describethe pumping of reagents to immobilized cells.

The danger of active movement is killing cells by rapid changes to theirenvironment. Cells can be trapped or injured by shearing forces producedby the rapid movement of cells or fluids moving to and past cells. Oneadvantage of active movement is being able to perform an operation ofcell manipulation rapidly. Another advantage is ability to have a rapidand fine control of the chemical environment surrounding the cells.These advantages also allow new manipulation and control of cells andreactions of cells with reagents. In addition, active movement allowsthe cells to be used as a stationary phase in a chromatographic column.

There are several types of cell active movement. These include:

-   1. Active movement of cells to and from a column, to the column    medium and to capture functional groups, all without damage to the    cells. This allows the opportunity of the column to capture live    cells in a reversible form.-   2. Active movement of reagents to live cells on the column to able    to perform chemical reactions and interactions with the live cells.    Rapid and fine control of the concentration of the reagents is    possible while removing other reagents that may have side reactions.    Even finer control is achieved with the use the cell-compatible, low    dead volume frits and impervious column media. Through control of    chemical or physical changes such as pH or concentration,    interrogation of reagents and cells is performed. Reagents and    interactions can be driven to completion rapidly and completely.-   3. Active movement and recovery of biological products from the live    cells on the column. The products may change depending on how the    cells are treated or by changing the materials that may contact the    cells. The products may be studied or used.-   4. Active movement removal of non-specific materials (background    cells and other nonspecific or undesired molecules) from cells of    interest. This removal it possible to accurately distinguish between    cells of interest and background cells to dramatically improve    purity, detection, chemical interrogation, chemical reactions, etc.-   5. Active movement of reagents to add and to remove analyte reagents    from cell stationary phase column. Analyte reagents can be added and    recovered rapidly from the cell stationary phase column for analysis    or further operations under controlled chemical elution conditions.-   6. Active movement of reagents to remove cells from column. Living    cells can be recovered rapidly from the capture column for analysis    or further operations under controlled chemical elution conditions.    Removal can be complete and rapid. Cells can be detected on line for    real-time quantification and interaction measurements.

Active movement is rapid but gentle and can help cells to remain alive.Living cells can be tagged on column under controlled chemicalconditions. Living cells and reagents that interact with living cellscan be interrogated on column under controlled chemical conditions.

Another aspect of active movement for living cell columns is the preciseand accurate control flow of reagents through the column. With columnsof the invention, the concentration of the reagents flowing to andflowing from the cells on the column is controllable and predictable.One means of being able to accomplish this using low dead volume fritsto hold the media in the column that does not contain dead spaces orspaces that trap liquids or materials. Another means of being able toaccomplish this is by using media that is impervious. This means reagentconcentrations are not diluted or changed unpredictably in concentrationby penetration of reagents or cells into the media matrix. These changesin reagent concentration by penetration are explained in a latersection.

Dilution Prevention for Reagents and Eluents

In some embodiments of the invention, reagents are introduced into acolumn in a unidirectional flow. Introduction of a reagent into a columnforms the wave front of the reagent traveling through the column. It wasdiscovered that for some embodiments of the columns of the invention,the wave front for an eluent slug traveling through the column wouldtravel at slower speed than expected. Furthermore, the shape of the wavefront was diffuse and the width of the wave front was broad. Cells couldnot be eluted quickly and effectively. Then, it was discovered that thetime necessary for adding reagents to a column was also slower thanexpected. For example, the process of adding an antibody capable ofcapturing a cell required an appreciable time to react all of thefunctional group sites. Also, eluting a captured cell from a columnrequired a large amount of eluting reagent and an appreciable amount oftime and flow to effectively elute cells from the column. Performing thereaction by adding reagents in a unidirectional flow can be slow due toslow reaction kinetics. But it was discovered that an additional factorappeared to contribute to the slow reactions.

It was discovered that some reagents would enter the interior of thebead whereas the cells would penetrate very little or remain on thesurface of the bead. Because of this, reagents were being used orconsumed in locations within in the columns where cells were notpresent. Thus, some reagents were consumed but did not serve any usefulfunction with regard to capture of cells, reacting with cells or releaseof cells.

It was further discovered that this uneven distribution of reagentsdepended on the type and size of reagent. Very large materials such ascells or biomolecules such as DNA, RNA or other (bio) polymers); smallermaterials such as biomolecules including antibodies, fragments ofantibodies, lipids, nucleic acids, aptamers; smaller materials such asbuffer reagents including organic compounds, salts, bases, and acids;and finally very small materials such as the molecules of the solventitself water all entered or penetrated columns of the invention todifferent degrees and depths, depending primarily on the size of thereagent and property of the substrate. Furthermore, it was discoveredthat when a solution containing these materials was pumped into acolumn, the localized concentration of some reagent materials wouldchange depending on whether or not the material entered the matrix ofthe column media. If the reagent entered the bead matrix, then thelocalized concentration of the reagent would decrease, especiallyrelative to a reagent that did not enter the resin matrix. The degree towhich the concentration decreased depended on the degree the reagentcould enter the resin phase. The smallest reagents were diluted mostwhile intermediate reagents were diluted to a smaller degree and thelargest reagents, cells, normally did not decrease the localizedconcentration of the material introduced into the column.

The reagent concentration and amount needs to be considered carefullyfor each step of the purification method including 1) activating acolumn for capture, 2) capturing a cell from solution 3) washingnon-specific materials away from a column containing captured cells, 3)reacting a tag to cells contained within or captured by a column, 4)washing away unreacted tagging reagent from a column, 5) introducing aneluting reagent to release captured cells from a column, 6) introducingreagents to controllably lyse cells captured by a column, 7) introducinganalyte reagents to a column containing a cell stationary phase and 8)eluting or displacing analyte reagents from a cell stationary phasecolumn.

It was discovered that the concentration profile of a material pumpedthrough a column is a wave front that travels through the column as itis being pumped. The concentration profile of reagent that penetratesthe resin matrix will decrease and be lower than the concentrationprofile of a reagent that does not penetrate the bead. That is, the wavefront is delayed if the reagent penetrates the column medium.

Two methods for controlling the localized reagent concentration ofreagents introduced to columns of the invention were developed. For theconsideration of this discussion, the term reagent may refer to verylarge materials (e.g cells, DNA, RNA), larger materials (e.g.antibodies, fragments of antibodies, lipids, nucleic acids, aptamers)smaller materials (e.g. buffer reagents, organic compounds, salts,bases, and acids) or very small materials (e.g. solvent molecules). Thefirst method involves changing the reagent concentrations relative toeach other depending on whether or not a reagent (or material ormolecule type) penetrates the resin matrix. If any particular reagentpenetrates the resin, then the concentration of that reagentconcentration is increased in correspondence to the degree ofpenetration into the resin. That is, for reagents that penetrated theresin matrix, the concentration of the reagent can be increased tocompensate for dilution of the reagent. For a fully penetrating reagent,the concentration can be increased by up to almost 100% because theresin matrix occupies approximately 50% of the column volume. If thereagent only penetrates 50%, then the concentration of reagent would beincreased 50% to maintain the localized concentration relative to anon-penetrating reagent.

A second method is to control and limit the penetration of reagents andsolvent into regions of the media where cells do not reside. One way toaccomplish this is to use a resin impervious to the specified reagent inthe same manner that cells are impervious. For example, in certainembodiments, a resin can have very small pores that don't allow entry ofmost reagents. In some embodiments, the resin is impervious.

In still other embodiments, the resin contains large pores, big enoughfor cells to reversibly enter. If it is desired to control or increasethe concentration of any particular reagent as the wave front is passedthrough the column, then the column solid phase is chosen to be orreacted to be made impervious to that particular reagent.

The cell penetration of a media substrate can be less than 10%, lessthan 5%, less than 1% or is none or zero penetration into the imperviousresin matrix. The reagent penetration into a medium substrate can beless than 50%, less than 20%, less than 10%, less than 5%, less than 1%or it is possible to have no penetration when using an impervious resinmatrix. Reactions in which controlling reagent penetration into thesubstrate should be considered include 1) activating a column forcapture, 2) capturing a cell from solution 3) washing non-specificmaterials away from a column containing captured cells, 3) reacting atag to cells contained within or captured by a column, 4) washing awayunreacted tagging reagent from a column, 5) introducing an elutingreagent to release captured cells from a column, 6) introducing reagentsto controllably lyse cells captured by a column, 7) introducing analytereagents to a column containing a cell stationary phase and 8) elutingor displacing analyte reagents from a cell stationary phase column andother reagents used in a cells purification, cell detection and cellstationary phase chromatography.

Affinity resins have a gel like, hydrophilic structure that swells inthe presence of water or polar solvents. The swollen polymers containpores that allow solvent to diffuse in and out of the resin bead. Theswelling can be significant. For example a cellulose, agarose orSepharose substrate will swell 5-10 times its original size whencontacted with water. In the swelling process pores are opened upproducing beads with a pore diameter up to 500 angstroms and largerallowing biomolecules to migrate and diffuse into the bead along withthe solvent.

In some embodiments of the invention, a polymer substrate is used thatdoes not swell upon exposure to water. In substrates which do not swellin water (solvent), buffer molecules, bio molecules and/or cells cannotenter pore in the substrate. The substrate may be polystyrene,polyacrylate type, polyester, or other olefin polymer or other polymer,or inorganic substrate material. Inorganic polymers include polysiloxaneand polyphosphazene, silicone, etc. Inorganic materials include aluminumoxide, zirconia, silica, etc. Organic polymers include low densitypolyethylene (LDPE), high density polyethylene (HDPE), polypropylene(PP), polyvinyl chloride (PVC), polystyrene (PS, nylon, nylon 6, nylon6,6, Teflon (Polytetrafluoroethylene), thermoplastic polyurethanes(TPU), polytetrafluoroethylene (PTFE), polychlorotrifluoroethylene(PCTFE) and other polymers. When exposed to water, the particle sizeincrease of these substrates is less than 5%, 4%, 3%, 2%, and 1%.Swelling may be controlled by controlling the polarity of the interiorof the substrate to be nonpolar or non-hydrophilic. Water is limited inentering the interior of the bead and hydrating the bead.

In other embodiments of the invention, the swelling of the substrateupon exposure to water may be more, but still limited. In these casesthe interior of the bead or substrate may be more hydrophilic, butswelling is limited because the polymer beads are crosslinked or heldphysically together. In some embodiments water may not enter thesubstrate. Buffer molecules, biomolecules and cells also may not enterthe substrate. In some embodiments, some limited amount of water andbuffer molecules may enter the substrate, but bio molecules and cellsmay not enter the substrate. When exposed to water, the particle sizeincrease of these substrates is less than 50%, 40%, 30%, 20%, 10% 5%,4%, 3%, 2%, and 1%.

Another counterintuitive method for increasing the effectiveness ofreagents was developed for columns of the invention. Consider a columnin which cells are connected to a streptavidin resin via a biotinylatedantibody. Cells can be eluted from such a column with biotin in a firstorder replacement reaction. First order means that there is a one-to-onereplacement of the eluent molecule to the biotinylated antibody holdingthe cell to the media. The reaction is an equilibrium reaction. Theequilibrium of the reaction shifts as the biotin concentration isincreased.

However, another way to increase the effectiveness of the reaction is tolower the capacity of the resin. With a lower capacity resin, a lowerconcentration of biotin can be used to drive the equilibrium reaction tosame extent. In a related manner, if the same biotin concentration isused in a pervious higher capacity resin versus a less pervious lowercapacity resin, then the lower capacity resin will respond faster. Thelower capacity resin will also respond to a greater extent with the sameconcentration of biotin because the equilibrium reaction will be shiftedto more complete reaction. That is, a reaction can be driven tocompletion by adjusting either the resin capacity or the reagentconcentration. More complete elution or displacement of the biotinylatedantibody occurs with the effectively higher amount of reagent. Also, ifthe reagent is consumed in the reaction, as they are in displacementreactions, then the mass amount of the reagent needed is also lower withthe lower capacity of the resin.

In another embodiment, an antibody attached to a cell is bound to aprotein A resin. The antibody can be displaced by competition with aprotein A molecule.

Finally, there is another factor that controls the amount of eachreagent needed and the effectiveness of reagents added to the column.The actual capacity of the resin is determined by the actual number offunctional groups in a resin. This can be expressed for example asmilliequivalents of functional group per mL of bed volume of a column ormilliequivalents of functional groups per gram of resin, etc. But theeffective capacity of a resin can be much different and often lower thanthe actual capacity. The effective capacity of the column is a measureof sites that function in capturing, processing, using and recoveringcells in the column. The effective capacity does not include functionalsites that do not capture cells. In some columns of the invention, theratio of actual capacity to effective capacity is quite high becausemost of the sites are not accessible to the cells. In the columns of theinvention, the ratio of actual capacity to effective capacity can be inthe range of 1000 to 1 or 100 to 1. However, if the column does notcontain functional groups that are impervious to cells, then the ratiois much lower, which can be desirable. In some columns of the invention,the ratios are much lower and require lower reagent concentrations.Desired ratios of actual capacity to effective capacity for cells forcolumns of the invention include 10 to 1, 5 to 1, 3 to 1, 2 to 1, 1.5 to1, 1.2 to 1 and is 1.1 to 1, 1.05 to 1 and 1 to 1.

In some embodiments, a more impervious resin is beneficial. By limitingplacement of the functional groups to sites that are accessible to thecell, two things are accomplished. First the capacity per unit volume ormass of the column is lowered making the reagents introduced into thecolumn more effective per unit concentration. In some embodiments,improved columns of the invention have capacities in the range of100,000 cells per mL of column bed volume, 500,000 cells per mL, 750,000cells per mL, 1,000,000 cells per mL, 2,000,000 cells per mL, 5,000,000cells per mL, 10,000,000 cells per mL, 20,000,000 cell per mL,50,000,000 cells per mL, 100,000,000 cells per mL, 200,000,000 cells/mLor 500,000,000 cells per mL of column bed volume. Second, the ratio ofactual capacity to effective capacity for cells becomes lower. This alsoincreases the effectiveness of reagents introduced into the columnbecause they are interacting only with functional group sites that areaccessible or useable by cells. In some embodiments, this means placingor having the sites on the surface of the bead or surface of the media.Still, from a standpoint of recovering as many purified cells aspossible, it is desirable to increase the column capacity to as high aspossible. One way of accomplishing this is to increase the total surfacearea of a column by decreasing the bead diameters contained in thecolumn. However, increasing the capacity by decreasing the bead size maynot be possible because this may also increase the restrictions to flowwith smaller spaces between the resin beads. Therefore, although it iscounterintuitive, low capacity resins of the invention are moredesirable for this reason as well.

Temperature

In some embodiments, the column is operated in a cold room while inother embodiments, the column can be operated at room temperature or ata temperature greater than room temperature. The optimum temperature forrunning the column will depend on parameters such as the application,the column medium and the cell type. In some embodiments, the columnsare operated in a hood such as a laminar flow hood to maintainsterility.

-   1. Temperature may be used to help preserve cells. These can be    sample cells, cells captured on the column or cells eluted from the    column.-   2. Temperature may also be used to change the nature of the cells    being captured and control selectivity of the column to capture    particular cells.

For example incubation of red blood cells at 37° C. may affect theexpression of some antigens, such as CD35 and CD11b. While temperaturemay not affect expression of CD15s, CD44, or CD62L, maintaining atemperature of 37° C. may accelerate apoptosis of neutrophils withsubsequent shedding and decreased expression of CD16. Peripheral bloodneutrophils prepared at 4° C. may express less CD35 and CD11b than thoseprepared at room temperature, suggesting release of some granularcontents at higher temperatures. It has been found that changes insurface marker expression of CD11/CD18 occurred with temperature change,such as warming neutrophils from 4° C. to 37° C.

In some embodiments, the sample and the column are maintained at a lowertemperature such as 4° C. to retain the integrity of the sample andmaintain a constant selectivity. In some embodiments, the sample and/orcolumn are maintained at a higher temperature up to 35° C., 37° C., 40°C., 45° C., 50° C. and even higher. In some embodiments, the sampleand/or column are maintained at sub-ambient temperatures or higher thanambient temperatures depending on the particular property studied orused.

Capture and Elution Strategies

Various mechanisms can be used for cell capture on the medium.Non-limiting examples include a functional group that has affinity forthe cells, use of a tagged antibody, ion exchange, a tagged aptamer andan antibody loaded resin (Pro A, G etc.) covalent bonded linkers (alkylthio, etc.), hydrogen bonded linkers. In some embodiments, abiotinylated antibody binds a cell surface marker and cells are isolatedusing a streptavidin resin. In certain embodiments, the resin can becomprised of an antibody. Other capture mechanisms such as hydrophobicinteraction, reverse phase, normal phase, ion pairing and ion exchangecan be used as long as the cells are not damaged.

Antibodies used with the invention can bind cell surface markers. Thereare many commercially-available antibodies that bind cells. One list ofover 2800 antibodies can be found using the product finder on theMiltenyi Biotec website.

The following is a non-limiting list of human cell surface markers thatcan be identified by PCR (see the SABiocsiences website).http://www.sabiosciences.com/rt_per_product/HTML/PAHS-055A.html). Listsare also available for mouse and rat cell surface markers.

B-Cell Surface Markers:

Activated B-cells: CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2,IL2RA, TNFRSF8, CD70 (TNFSF7).

Mature B-cells: CD19, CD22, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B,CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1.

Other B-cell Surface Markers: CD1C, CHST10, HLA-A, HLA-DRA, NT5E.

T-Cell Surface Markers:

Cytotoxic T-cells: CD8A, CD8B.

Helper T-cells: CD4.

Activated T-cells: ALCAM, CD2, CD38, CD40LG, CD69, CD83, CD96, CTLA4,DPP4, HLA-DRA, IL12RB1, IL2RA, ITGA1, TNFRSF4, TNFRSF8, CD70 (TNFSF7).

Other T-cell Surface Markers: CD160, CD28, CD37, CD3D, CD3G, CD247, CD5,CD6, CD7, FAS, KLRB1, KLRD1, NT5E, ST6GAL1.

Natural Killer (NK) cell Surface Markers: CD2, CD244, CD247, CD7, CD96,CHST10, IL12RB1, KLRB1, KLRC1, KLRD1, NCAM1.

Monocyte and Macrophage Cell Surface Markers:

Activated Macrophages: CD69, ENG, FCER2, IL2RA.

Other Monocyte and Macrophage Surface Markers: C5AR1, CD163, CD40, CD63,CD74, CD86, CHST10, CSF1R, DPP4, FCGR1A, HLA-DRA, ICAM2, IL1R2, ITGA1,ITGA2, S100A8, TNFRSF8, CD70 (TNFSF7).

Endothelial cell Surface Markers: ENG, ICAM2, NOS3, PECAM1, SELP, TEK,VCAM1, VWF.

Smooth Muscle cell Surface Markers: MYH10, MYH9, MYOCD.

Dendritic cell Surface Markers: CD1A, CD209, CD40, CD83, CD86, CR2,FCER2.

Mast cell Surface Markers: C5AR1, FCER1A, FCER2, TPSAB1.

Fibroblast (Stromal) Surface Markers: ALCAM, COL1A1, COL1A2.

Epithelial cell Surface Markers: CD1D, KRT18, KRT5, KRT8, EPCAM.

Adipocyte Surface Markers: REIN.

One strategy involves competition. Cells are captured with a ligand thatbinds a cell surface marker and then eluted with the same ligand. Inanother example, cells bound to antibodies captured on ProA resin can beeluted with ProA or a similar molecule. Alternatively, the ligand couldbe bound to a tag which in turn, binds an antibody as shown in FIG. 4.

Another competition strategy utilizes ANTI-FLAG resin. A FLAG-labeledFab or Antibody that binds a cell surface marker could be engineerede.g., in E. coli. Many other types of functional groups can be used forcompetitive, equilibrium type reactions to capture and optionally eluteand recover cells.

Alternatively, cells can be eluted by a physical change such as a changein pH or temperature as shown in FIG. 5. Preferably, an eluent can beselected that does not harm the cells, particularly when the recovery ofviable cells is desired. In one example, a temperature-sensitive ProAresin can be used such as Byzen Pro resin made by Nomadic Bio Science.Using this type of resin, cells can be eluted at neutral pH byincreasing the temperature as shown. In a second example, cells can becaptured by antibodies specific to cell surface markers and eluted usinga low-pH eluent. In this example, the elution step could be performedrapidly followed by a quick transfer of the purified cells to aneutral-pH solution.

A variety of affinity strategies can be used to capture cells on thecolumn. However, it is also possible to use ion exchange. In alternateembodiments, cell capture may not be desired. Gel filtration(size-exclusion chromatography) can be used to enrich a particular celltype by separating cells away from non-cell components or by separatingcells from each other based on their size. For example, circulatingtumor cells (CTCs) are larger than other cell types and can be separatedfrom other cells using size exclusion chromatography. Gel filtrationcould also be used to clean up a sample, e.g. a diagnostic sample.Non-cell material could be removed or taken up by the column.

In some embodiments, cells captured on a column can be eluted usingenzymatic cleavage. For example, cells could be captured using proAresin charged with antibodies that bind a cell surface marker. Theantibody could then be cleaved with an enzyme such as papain or pepsinto elute the cells.

The columns and methods of the invention can be used to capture andelute viable healthy cells or diseased cells. In certain embodiments,cells can be captured using an aptamer specific to a cell surfacemarker. Aptamers can be single- or double-stranded RNA or DNAoligonucleotides. Aptamer sequences can be determined using SystematicEvolution of Ligands by Exponential Enrichment (SELEX) or otherselection processes (see for example Base Pair BioTechnologies, Inc.,Houston, Tex.). The aptamers can contain non-standard or modified bases.As used herein, a “modified base” may include a relatively simplemodification to a natural nucleic acid residue, which confers a changein the physical properties of the nucleic acid residue. Suchmodifications include, but are not limited to, modifications at the5-position of pyrimidines, substitution with hydrophobic groups, e.g.,benzyl, iso-butyl, indole, or napthylmethyl, or substitution withhydrophilic groups, e.g., quaternary amine or guanidinium, or more“neutral” groups, e.g., imidazole and the like. Additional modificationsmay be present in the ribose ring, e.g., 2′-position, such as 2′-amino(2′-NH₂) and 2′-fluoro (2′-F), or the phosphodiester backbone, e.g.,phosphorothioates or methyl phosphonates.

Aptamers have been shown to be capable of cell capture. For example,Shen et al. used DNA aptamer-functionalized silicon nanowires to captureand release non-small cell lung cancer cells (Shen, et al. AdvancedMaterials, Volume 25, Issue 16, pages 2368-2373, Apr. 24, 2013).

Aptamers can be chemically conjugated to chromatographic beads. Forexample, see Zhou et al., Trends in Analytical Chemistry. 2012 41:46-57.Alternatively, biotin-labeled aptamers could bind streptavidin resin.Cell elution can be performed by a means with disrupts the aptamer orthe aptamer-cell bond. For example, RNase could be used to performelution from an RNA-based aptamer as shown in FIG. 6. Other elutionstrategies that can be employed with aptamers are anti-sense,photocleavage (at an appropriate wavelength), use of an enzyme, heat,denaturing solution or chemical cleavage. An aptamer comprised of adisulfide bond could be treated with a reducing agent to disrupt thebond and release a bound cell. An aptamer containing amagnesium-dependent fold could unfold and release a bound cell with theaddition of a chelator.

The capture of cells is difficult, especially under flowing stream,especially under high flow rates, especially under high linear velocityof fluid containing cells traveling through the column.

Cell surface markers can be used to capture the cells. However differentcell surface markers may have different uses or functions in methods ofthe invention. For example, a cell may be captured with one type ofmarker and tagged with another type of marker. A third marker or moreand/or combination of markers may be interrogated, measured or studied.

Elution of the cell can be accomplished using a strategy directed towardrelease of the capture marker without disrupting any of the othermarkers. However, if the cell is used in a cell stationary column, thencapture and elution of analytical reagents can be performed with a thirdtype of marker, without disrupting any of the other markers.

Once the cell stationary phase/analyte reagent measurements areperformed, the cells of the stationary phase may be analyzed. A tag maybe introduced into the column to give a detectable signal to the cell.In these cases a suitable marker on the cell may be targeted to reactwith the cell to make the cell detectable. A tag may be introducedbefore cell elution or after cell recovery.

Removal of the Solid Chromatography Medium from the Column

In some embodiments, the resin with cells attached can be removed fromthe column after the capture and wash steps. In these embodiments, theresin (with cells attached) can be placed in a well. Cell lysis can beperformed if desired. PCR can be performed either on whole cells orlysed cells. Nucleic acids can be isolated and analyzed e.g., bysequencing.

Removal of the resin can be performed by piercing the bottom frit of thecolumn and then pushing the resin into a well with air or liquid. A fritpiercing tool can be used for this purpose. In some embodiments, thefrit piercing tool is comprised of a handle and a piercing pointhowever, a wide variety of geometries are possible. The tool can be usedmanually by grasping the handle and pushing the piercing point of thetool through the column frit and into the bed. Then the tool is removedand the column is placed above a tube or microplate well which willreceive the resin. Air or liquid can be used to push the resin into thewell.

In another embodiment, the frit piercing tool can be recessed in a well,handle side down into the well of a microplate. The column is positionedabove the well and pushed down into the well to pierce the frit. Thepiercing tool can be removed or remain in the well. Air or liquid can beused to push the resin into the well.

These embodiments can be performed in automated parallel fashion.

Large Column Operation

Large columns are defined herein as those columns having a bed size ofat least 100 μL and a capacity between 2 mL and 100 mL. In someembodiments, large columns can be pipette tip columns while in otherembodiments, the large columns are not pipette tip columns. Largecolumns that are not pipette tip columns have a body and bed volume of1-100 mL.

Larger columns of the invention have a number of different propertiesfrom the smaller columns. It is not simply a matter of scaling up smallcolumns to produce large body affinity columns. First, larger columnscan have a different geometry than the smaller columns. Specifically,the ratio of the column diameter to the resin bed height can be greaterin the large columns.

Second, it is possible to use smaller liquid volumes relative to the bedvolume. For example, in the smaller, previously-described columns aminimum of 2 bed volumes could be aspirated. But in the larger columns,it is possible to aspirate one bed volume of liquid.

Furthermore, higher flow rates can be used with the larger columns.Columns that have a body size of at least 2 mL and a bed volume in therange of 200 μL to 50 mL can be operated using significantly faster flowrates than columns having a smaller column body and bed volume. Flowrates in the range 1 mL/min to 12 mL/min and faster can be used. Inlarge column embodiments, the flow rate for passing liquids through thecolumn can be within a range having a lower limit of 0.5 mL/min, 1mL/min, 1.5 mL/min, 2 mL/min, 2.5 mL/min, 3 mL/min, 3.5 mL/min, 4mL/min, 4.5 mL/min, 5 mL/min, 6.5 mL/min, 7 mL/min, 7.5 mL/min, 8mL/min, 8.5 mL/min, 9 mL/min, 9.5 mL/min, 10 mL/min, 10.5 mL/min, 11mL/min, 11.5 mL/min, 12 mL/min or greater. The upper limit of the flowrate can be in the range of 60 mL/min, 70 mL/min, 80 mL/min, 90 mL/min,95 mL/min or 100 mL/min.

As a result of the higher flow rates, the separation times are shorterand cells can be isolated and recovered in a very short time. In someembodiments, cells can be isolated from a biological sample in less than45 minutes, less than 30 minutes, less than 25 minutes, less than 20minutes, less than 15 minutes, less than 10 minutes or even less than 5minutes.

On-Column Lysis or Interrogation

In certain embodiments, the cells are not eluted but instead aremanipulated or interrogated on the column. The cells can be labeled oncolumn, for example with a fluorescent antibody or aptamer. Cells can belysed on column and cell components (e.g., nucleic acids) can be elutedand analyzed.

In some embodiments, the cells are not eluted from the column. Insteadthe cells may be studied or used while on the column. The cells can belysed on column or the column bed material with cells bound can bereleased from the column and subjected to further analysis such as apolymerase chain reaction. Nucleic acids, DNA or RNA associated withinwith the cells or that have bound to the cells or released from thecells may be measured.

In some cases it is desirable to label cells on the column for instancewith an antibody, fluorescent tag or aptamer. On-column labeling can beuseful for diagnostic applications by enhancing the signal to noiseratio.

In some embodiments, the columns of the invention can be used todistinguish live and dead cells. Reagents sold by Life Technologies andothers can be useful for these methods.

On column lysis can be done slowly and with large volumes of lysissolution so that the column is not plugged.

Cell Therapy

The invention additionally includes devices and methods for treatingdiseases. In some embodiments, healthy cells can be transferred betweenorganisms. For example, donor cells from a healthy pancreas can beisolated on a column and transplanted into a patient suffering from Type1 Diabetes. Stem cells are used for bone marrow transplantation willlikely have a variety of therapeutic applications in the future.

Stem cells and other cells may be captured, purified with packed bedcolumn technology in an open system or a sealed system and thenmanipulated using CRISPR type genome editing methodologies andtechnologies. The cells may be collected for further downstreamprocessing.

Crispr technology uses the cas9 enzyme with a guide RNA sequence tocleave and edit the genome at specific site while in the cell. Thistechnology allows the researcher to specifically cut and insert/edit thegenes at will. One of the major applications for this is gene therapy.Disease caused by one gene error can be fixed using Crispr to fix thegene to cure the disease. Another application is the manipulation ofstem cells or early stage cells. Defective genes may be edited tocontrol or eliminate disease or other genetic factors.

There are many research applications. Knock out organisms (knock outmice) can be created to facilitate researching genetic pathways. Similarknock out work can be performed with cell lines to create for examplecell lines that function to perform specific things. You can createdifferent strains of plants can be created by changing/editing the genesto create resistant strains, more fertile strains or strains withspecific attributes.

Editing of the cells with Crispr technology may be performed while thecells are captured on columns of the invention or after they have beeneluted and recovered from a column of the invention.

Samples containing donor cells can be obtained from a variety of sourcesincluding human, animal and cell culture. For example, donor cells canbe obtained from cell culture, body fluids such as blood or lymph, organtissue, bone marrow, etc. Donor cells can be engineered cells.

In other embodiments, cells are used to deliver gene therapies. Genescan be introduced into cells for example, by using areplication-defective adenovirus to produce engineered cells. Thesecells can perform a variety of therapeutic tasks such as deliveringdrugs, destroying cancer or regulating the immune system.

In one study, T cells were engineered to produce antibodies that bindcancerous cells (Grupp et al., N Engl J Med. 2013 Apr. 18;368(16):1509-18). These engineered T cells were introduced into patientswith leukemia to achieve remission or tumor size reduction. In this typeof application, patients' T cells could be isolated using a column ofthe invention, engineered and then proliferated in cell culture. Afterthe engineered T cells were grown, they could be isolated with a sterilecolumn prior to introduction into a patient.

The columns and methods of the invention can be used to isolate cellsused for cell-based therapy. In these embodiments, sterile columns canbe used for cell isolation. One advantage to this approach is that thecell populations obtained will be free of contaminants. In certainembodiments, cells isolated from columns are administered to patients totreat diseases such as cancer, diabetes, heart disease, Parkinson's,Alzheimer's, liver disease and others. Healthy cells can be used toreplace cells in damaged or diseased organs. Cells isolated from anysource can also be transferred to a different individual or organism.For instance, cells can be transferred to a mouse or other animal modelfor research, diagnostic, characterization or medical purposes.

Organ Cells on a Column.

Cells isolated from any source can also be transferred to a differentindividual or organism and contained on a column. Cells contained incolumns and the products from columns may be used for research andmedical purposes. They may be interrogated chromatographically todetermine what interacts with the cells and the manner in which theyinteract. These experiments may be performed in parallel or serially.The effects on the cells may be studied. The biological productsreleased from the cells may be used for research and medical purposes.

Columns containing captured cells of specific organs can be used fordrug testing. For example a column containing liver cells can be used tostudy the interactions and the effects these interactions of drugcandidates with liver cells infected with hepatitis B, a viral infectionof the liver.

Columns of the invention can capture kidney cells, intestine cells,muscle, cells, fat cells, bone cells, skin cells, pancreas cells, bloodvein cells, etc. Furthermore, columns containing different organ cellscan be studied together. For example, a drug candidate designed tointeract with prostate cells might also interact with liver cells insuch a way that might produce toxic products. The flow from columns maybe collected and analyzed with mass spectrometry, infrared spectrometry,UV spectrometry or other analytical tools. Columns of the inventioncontaining for example, parotid gland cells of the mouth or Eccrinesweat gland cells of the skin can be used to measure the interaction ofseveral molecules. These molecules can be contacted with columns of theinvention containing other cells such as liver cells, or kidney cellsfor example and the interactions and effect of interactions measured.The fluid that is pumped through the column or columns of the inventioncan be blood-mimicking fluids which bring sustenance to the cells.

Columns of the invention may be used to capture human inducedpluripotent stem cells, adult stem cells that are treated to becomeembryonic state and then encouraged to become many different types oftissue. The use of stem cells on columns of the invention can be used torepresent an individual. In this way, columns representing variousorgans could be derived from a single person. Or cells obtained from abiopsy from an individual can be captured onto columns of the invention.Experiments could then be carried out on the various columns todetermine the interactions and effect of interaction of drugs at variousconcentrations or amounts (dosages), or combinations of drugs at variousratios, concentrations or amounts.

Therapeutics Screening

Columns and methods of the invention can be used to screen drug leadsand identify drug targets. Disease cells can be immobilized on thecolumn medium and challenged with pools of drug candidates such as smallmolecules, engineered proteins (such as engineered T-cell receptors),biologics or other entities. The column can be washed to remove speciesnot tightly bound.

At this stage, the cells bound to drug candidates can be interrogated.Different solvent conditions can be applied to the column to testbinding and dissociation conditions. Alternatively, cells bound to adrug candidate or other entity can be released from the column andstudied. For example, cells can be disrupted to create membranefragments consisting of cell surface components bound to drug targets.The drug targets and the drug leads can be identified using methods suchas mass spectrometry.

Alternatively, drug candidates can be immobilized on a column anddifferent cell types can be passed through the column to identify andthen characterize interaction. In some embodiments, cells can bemanipulated prior to passing them through the column. For example, cellscan be mixed with a drug candidate and subjected to competitionexperiments with other drug candidates present on the column.

Cell Clean-Up

The columns and methods described herein can additionally be used forcell clean-up. For instance, it can be desirable to separate cells fromcontaminants, collect materials from cell populations or perform bufferexchange. Existing methods for cell clean-up include magnetic beads,dialysis and centrifugation which are time-consuming, single equilibriumprocedures. The columns and methods of the invention provide a rapidalternative and offer the advantage of being a multi-equilibriumprocess.

In some embodiments, cells are purified away from contaminants bycapturing contaminants on the column while cells pass throughunencumbered. Contaminants can be captured on the solid phase using forexample, an affinity group, aptamer capture, ion exchange or otherstrategies. Alternatively, size exclusion can be used to separate cellsfrom contaminants. Using size exclusion, cells might pass through thecolumn quickly while smaller contaminating molecules might enter thesolid phase which would cause them to pass through the column moreslowly.

Diagnostics

Some requirements of a good diagnostic procedure are that it is rapid,simple, enriches the sample before detection, removes nonspecificmaterials (that could give a signal), high sensitivity, high signal tonoise, and linear signal.

The columns and methods of the invention can be used for a number ofdiagnostic applications including oncology, virology and infectiousdiseases. Diagnostic applications include isolation of any cell type andthe option of additional cell characterization on column or post column.One application is the identification of pathogens such as viruses,bacteria, fungi and protozoa from a patient sample. Another applicationis the isolation and characterization of cancer cells, such ascirculating tumor cells (CTCs) as described below in Example 3.Isolation of CTCs is useful for early cancer detection, characterizationof tumor cells, monitoring disease treatment, monitoring progression orremission.

Diagnostic applications of the invention can be used in a variety ofsettings. In certain embodiments, diagnostics are utilized in a researchsetting such as academia, biotechnology or pharmaceutical company. Inother embodiments, the columns and methods of the invention can be usedin point of care settings including emergency rooms, intensive-careunits, patients' bedsides, physician's offices, pharmacies and bloodbanks. In still other embodiments, diagnostic applications can comprisein-home tests. Diagnostics are also useful in a corporate setting suchas the food industry.

Diagnostic target cells can be any cell type listed above. As describedabove, cells are not defined herein as limited to entities capable ofself-replication. Included in the definition of cells are viruses,parasites and exosomes and organelles. A non-limiting list of diagnostictargets include mammalian cells, human cells, cancer cells, circulatingtumor cells, viruses, bacteria fungi and parasites. A non-limiting listfollows: Shigella, Salmonella, E. coli, Helicobacter pylori,Campylobacter, Chlamydia, Gonococcus, Streptococcus, Staphylococcus,Mycoplasma, Trichomonas vaginalis, Clostridium botulinum, HIV, HepatitisA, B and C, Herpes, Amoeba/parasites, Entamoeba histolytica,Acanthamoeba and Naegleria, Cryptosporidium, Giardia, Fungi such asCoccidiodomycosis (Valley Fever), blastomycosis, histoplasmosis,yeast—Candida albicans and other Candida sp. (hospital infections, bloodinfections) and opportunistic pathogens such as Cryptococcosis andAspergillosis.

Although it is not required for all diagnostic applications, a label canbe employed. For example, cells can be captured and then labelled on thecolumn. Alternatively, cells can be labelled prior to column capture.When cells are labelled prior to capture, the label can aid in cellcapture. In some embodiments, the label can actually be the entitycaptured on the column. Labelled cells captured on the column can bewashed, eluted and a detection step performed. In another embodiment,cells can be labelled following elution from the column. An advantage tothis approach is that a homogeneous cell population can be obtainedprior to the labelling step.

In some embodiments, only a few or relatively few of a particular cellsurface markers are used to capture a cell on the medium. For thisexample, we will call these markers Type A. This leaves most of thecaptured cell markers Type A on the rest of the cell untouched. Thelabeling of a cell can be performed by reacting these remaining, excessType A cell markers. In other embodiments, the labeling of a cell isperformed by reacting a cell marker, Type B, which is not used forcapturing the cell on the column. This strategy is especially useful foron-column tagging or labelling of the cells. By targeting a differentcell marker for labeling, the cell is less likely to be removed oreluted in the tagging process.

In some embodiments, the tagged cells are eluted and then detected. Insome embodiments, the elution of the tagged cell is performed bychanging the chemical nature of the linking or capture reagent.

A label is defined herein as any entity that can aid detection. Avariety of labels can be used for this purpose. For example afluorescent dye-labelled antibody or Fab can be used. In addition, anantibody or Fab can be conjugated with any kind of tag that aidsdetection. In addition to dyes, non-limiting examples of tags includeradioactive labels, proteins, enzymes (e.g., horse radish peroxidase),and metals including rare earth metals. Labels are not limited to taggedantibodies or Fabs; they include anything that can bind the cell surfacesuch as a protein, a dye or other molecule.

Dye-labelled antibodies or Fabs can be used to label specific cellsurface markers and viable versus dead cells. Dyes used to labelproteins include Ellman's Reagent, Coomassie Blue, Lowry reagents andSanger's reagent.

Post-column label detection can be carried out using a number ofdifferent methods. For instance, detection can be done with flowcytometry, a microscopy, a spectrophotometry, mass spectrometry, acolorimetric reader, a protein assay or a nucleic acid assay.

In alternate embodiments, on-column detection can be utilized. On-columndetection can be performed for example, by reflectance (UV or visible),fluorescence, colorimetric detection (e.g., ELISA), chemiluminescenceand others.

Cell-Based Stationary Phase for Liquid Chromatography

Columns of the invention can be used to produce and use a stationaryphase comprised of cells, referred to herein as the cell-basedstationary phase. The cells can be attached to a substrate and used tomeasure the interaction of analytes with a cell-based stationary phase.The cell stationary phase may be comprised of live or active cells.Active is defined herein as a cell that is not only living, but canundergo biological processes while attached to the column medium.Samples are injected into the mobile phase and enter the column andinteract with the cell-based stationary phase. These interactions can beidentified and quantified. Retention data and column interaction datacan be collected and analyzed. In many cases, the liquid phase flowthrough the column is unidirectional however, in some cases,bi-directional flow may be employed.

Generally, cells that are placed on the cell-based stationary phase areviable however, it is not mandatory. Cells attached to affinity resinspacked into a chromatographic bed have surface groups of various typesthat can interact with analytes flowing through the column. These groupsinclude proteins including glycoproteins, carbohydrates, lipids, sugarsand other groups. The proteins may contain phosphate groups, glycans,etc. Analytes such as drug candidates or antibody-drug conjugates caninteract with these groups by pumping them or injecting them into aliquid phase flowing through columns. Examples of interactive entitiesinclude antibodies (e.g. from antibody libraries), antibody-drugconjugates, FABS, proteins, enzymes, sugars, nucleic acids, lipids, ionexchange and other groups. This interaction can be measured in differentways, depending on how the chromatography is performed and the kineticrate constants of the on/off interaction of the cells with thestationary phase. Properties of the cells can be analyzed after they areremoved from the column. For example, cells can be removed from thecolumn after chromatography to measure chemical properties of the cells.They can be stained to count the numbers of live cells and dead cells.

The types of substrate used to form the cell-based stationary phaseinclude affinity, ion exchange resins and others. In fact, any chemistrydirected to cell surface markers can be used.

With living cell stationary phase chromatography, the types ofinteraction of an analyte with the cell stationary phase can becharacterized. Types of measurement include the kinetics of interaction,the extent or magnitude of interaction, the relative selectivity of theinteraction and other parameters. The column may be used to separateanalyte materials that have different selectivity. For example, twodifferent analyte materials may be associated or bound to a stationaryphase. An eluent may be added to the column and one material may beremoved from the column faster or more easily than the second material.This difference in selectivity may be measured.

In one example, cell chromatography may be used to determine whichreagents interact with stem cells and the effect of that interaction onthe cells. Stem cells are loaded onto a column to make a living cellstationary phase. Then, various analytes are introduced into the columnunder controlled chemical and physical conditions. The experiments maybe performed in parallel. Using chromatography, it can be determined theextent of interaction of a reagent with the stationary phase and how theinteraction can be controlled. The relative affinity of two or moreanalytes can be measured. Then after the chromatography is performed,the cells may be eluted and analyzed to determine the effect of theinteraction on the stem cells.

Chromatography with a living cell stationary phase may be used toexamine interaction of a particular type of cell and drug candidates.Cancer cells from a particular patient may be loaded onto a column.Existing drugs may be introduced into the column and the chromatographicinteraction measured to determine if a particular drug, series of drugsor a mixture of drugs may be suitable for treatment of the patient.After exposure and chromatography measurement of the analytes, the cellscan be removed and analyzed to determine the effect.

The cells are preferably living and stable in flowing fluid through thecolumn. The cells are not sheared from the column beads or media by thestream of liquid flowing through the column. The cells remain alivewhile attached to the column and remain available for use as a liquidchromatographic stationary phase for at least 30 min, at least 1 hr, atleast 2 hrs, at least at least 3 hrs, at least 4 hrs, at least 5 hrs, atleast 6 hrs, at least 12 hrs, at least 1 day, at least 2 days, at least3 days, at least 4 days, at least 5 days, at least 6 days or evenlonger.

Analytes of various types are introduced to the column of the inventionwith a mobile phase fluid flowing through the column. The analytesinteract with the cell stationary phase to different extents dependingon the kinetics and selectivity of stationary phase to the analyte. Theextent of interaction is measured with individual analytes or undercompetitive conditions with two or more analytes in a matrix ofbackground conditions. In addition, analyte materials of interest mayinteract with the cell stationary phase and be captured or removed froma complex matrix solution.

There are two general methods to prepare the system for capture of thecells by the column. In one method, the column medium contains sitesthat interact with the surface of the target cells. The medium may havethe sites or may be conditioned to gain the sites that interact with thecolumns. An example of this is where the column contains nucleic acids,lipids, peptides, antibodies or Fabs that can capture cells throughsurface markers. In another example, the column contains chelating orion exchange sites which can interact directly with a cell or maycontain a reagent that interacts with cells. In another example, thecolumn contains aptamer sites that interact with the surface of thecells.

Another method may be to treat the cells with antibodies or otherreagent(s) that allow the cells to bind the column medium. For example,antibodies can interact with cells in solution. The cells attached toantibodies can be captured by a Protein A or Protein G column. Inanother example, each antibody molecule may contain another functionalgroup such as a biotin. The biotin entity on the antibody (which isattached to the cell) may be captured by a streptavidin resin in thecolumn.

Different protocols may be used to capture cells for purification. Inone embodiment, the resin bead is activated or may be activated with anentity to be able to capture cells. After activation, a sample may beprocessed to capture the cells in the first step of purification. Inanother embodiment, cells may be treated or activated and then capturedby flow cells through the column.

In order to produce the cell stationary phase columns, the column andsubstrate must have the same cell accessibility characteristics ascolumns used to capture and purify cells. That is, the cells areaccessible to chemical interactions. Cells are not trapped in deadspaces and cells are not damaged by the frits or resin. The cellstationary phase columns are characterized by low backpressure, low deadvolume, and very little dead space.

In one procedure, the cells are attached to the surface of the columnpacking resin and then the resin containing the cell stationary phase ispacked into a column. The cells can be attached to the resin substratein a slurry. This attachment step can also be accomplished in two ways.The cells may be activated with an antibody or other chemical entitythat in turn, can attach to the resin. Or, the resin substrate may beactivated with an antibody or other chemical entity that can in turn,allow attachment of the cells. The cells are then mixed with the resinand the cells attach to the substrate producing the cell stationaryphase. Once the cell stationary phase is produced, the resin is packedinto the column.

Resins can be activated to be able to capture cells to make a cellstationary phase. One example of this is to load an antibody ontoProtein A column resin beads. The antibody is selective for surfaceproteins on the cells. Alternatively, cells can be activated to be ableto attach to resin beads. An example of this approach is the attachmentof a His-tagged Fab to cells. After removing excess Fab material bycentrifugation, the cells can be introduced to an IMAC resin bead. Thecells attach to the beads through the His-tagged Fab.

A resin substrate (not containing the cells) is packed into a column.The resin bead substrate contains an affinity group that can capturecells. The cells are introduced into the column and the cells attach tothe substrate producing the cell stationary phase. This capture step canbe accomplished in several ways. For example, the cells may be activatedwith an antibody, aptamer or other chemical entity that in turn, canattach to the resin. In another method, the resin substrate may beactivated with an antibody or other chemical entity that can in turn,attach the cells.

In one example, a column was prepared by gluing a frit on one end,packing the column and then gluing a second frit on the top of thecolumn. A 37-micron pore, 60 micron thick Nitex screen frit was attachedto the end of an acrylic tube 0.750 inches long, 0.500 inch outerdiameter and 0.375 inch inner diameter. Packing was accomplished bystanding the column on a stand with deep-well plate beneath the columnthat allowed liquid to flow out of the lower end. An aqueous slurry ofagarose resin, 45-165 micron particle size, was transferred to thecolumn by pipette. The packing material was not compressed. Excessliquid drained away filling the column with resin. Additional slurry wasadded until the bed of the column reached the top of the column. A Nitexscreen of the same material used for the other frit was glued onto thecolumn end using a methylene chloride solvent.

Silicone tape was wrapped round the column to increase the diameter.Then, two 10 mL plastic syringe bodies and male luer connections werecut to the 1 mL volume mark and placed on the end of the column. Thecolumn body was wrapped with stretchable silicone tape to seal thecolumn body. Male luer connections were connected to the inside of clearflexible Tygon tubing 0.250 inch outer diameter to connect to theinjector and fraction collector.

In a second example, the cells are attached to the column. A His-taggedFab is pumped through the column loading the column completely with theFab. The Fab is selective for a surface protein on the HeLa cancer cellline. After washing the column, HeLa cells are pumped into the columnloading cells onto the surface of the stationary phase. The column nowcontains a HeLa cell-based stationary phase. A chromatographic column ina chromatographic system is shown in FIG. 7. A column comprised of acell stationary phase is shown in FIG. 8.

Once the cell stationary phase column is prepared, chromatography can beperformed. Cells captured on the column can be interrogated with groupsof compounds or analytes to identify those that bind cells with thedesired affinity. For instance, a library of compounds can be added tothe column to determine which materials in the library have an affinityfor the cells on the column. The sample containing the library is addedto the column. The column can be washed. The stringency of the wash maybe varied to control capture of library materials. Then, the column canbe washed at high stringency to elute the compounds or the cells withcompounds attached.

Analysis to determine the identity of the compounds may be performedwith mass spectrometry. The identity and concentration of the compoundsrecovered may be determined by liquid chromatography or massspectrometry methods. The concentration of the various compounds mayindicate the ability of the cells to capture a particular compound.

The interaction of different materials with the stationary phase ismeasured using various chromatographic techniques. The extent ofinteraction under different conditions may be measured. The identity ofmaterials interacting may be determined. The ability of a reagent tobind to the cell may be measured. This measurement may be performedrelative to an eluent reagent or may be performed relative to anotheranalyte reagent.

The type of chromatography that can be performed depends on the kineticrates of the analyte interaction with the cell stationary phase. Forrapid kinetic interaction of analytes with the column, partitioningchromatography may be performed. Partitioning chromatography isperformed using unidirectional flow. Measurement of the analyteinteractions may performed using retention times or related values suchas capacity factors. Partitioning chromatography is performed underisocratic and gradual gradient conditions.

Other types of chromatography that may be performed with rapid analytekinetic interaction with the cell stationary phase include step gradientchromatography, displacement chromatography and frontal/breakthroughcurve chromatography. Step gradient and displacement chromatography canbe performed with either bidirectional or unidirectional flow.Frontal/breakthrough curve chromatography is performed withunidirectional flow.

For slower analyte kinetic rates of interaction with the cell stationaryphase, step gradient chromatography, displacement chromatography orfrontal/breakthrough curve chromatography can be employed. Also in thesecases, step gradient and displacement chromatography can be performedwith either bidirectional or unidirectional flow. The step gradient maybe one step gradient, after an optional wash or multi step gradient.Frontal/breakthrough curve chromatography is performed withunidirectional flow.

Some interactions may be additive. For example, calcium may be added tomembrane channels. In this case, the kinetic rate of uptake would behigher than release and breakthrough curve measurement may be moreappropriate.

Partitioning chromatography may require an injection of a slug ofanalyte into a flowing eluent stream, provided the partitioning israpid. But large injection volumes can be employed if the selectivity ofthe analyte for the column is high. Breakthrough chromatography requiresa continuous uniform (injection) supply of analyte. Injection is at thetop or inlet of the column for partitioning or gradual gradientchromatography. Displacement chromatography generally requires a largeinjection ensuring that the stationary groups are displaced with theeluent. Injection can be at the top of the column or at the bottom ofthe column for back and forth flow.

The following steps are an example of a partitioning chromatographicprocedure using living cell stationary phase column.

-   1. Treat or activate column or cells to provide suitable conditions    for the column media to be able to capture cells.-   2. Load cells onto column from a flowing stream. Loading may be    performed in a unidirectional or bidirectional mode.-   3. Wash nonspecific bound materials from the column using a flowing    stream.-   4. Pump an eluent through the column and through the detector to    ensure that all material has been washed from the column and a    stable detection baseline is achieved.-   5. Inject a single or mixture of analytes into the column.-   6. Pump an eluent solution through the column and measure retention    of analyte or analytes.-   7. Optionally recover cells from column stationary phase after    exposure to analytes and analyze.-   8. Optionally lyse cells or elute the components of the cells for    analysis. The lysis step may be done partially, or over a long time    period using gentle conditions to remove components of the cell for    processing and/or analysis.-   9. Remove and recover cells for analysis of the cells and/or further    research and development processing.

Detection may be continuous or fractions may be collected and analyzed.Analyte measurements include retention time, capacity factor,selectivity coefficient and others.

The bidirectional flow step gradient and displacement chromatography canbe can be performed with the pipette, syringe, gas pressure/vacuumchamber, peristaltic pumps and living cell stationary phase columns ofthe invention. This type of chromatography can be operated with a manualor electronic controlled pipette or automated robotic liquid handleroperated as a single channel or multiple columns in parallel or up to 96channels are operated in parallel.

The following steps are an example of gradient chromatographic procedureusing living cell stationary phase column.

-   1. Treat or activate column or cells to provide suitable conditions    for the column media to be able to capture cells.-   2. Load cells onto column from a flowing stream. Loading may be    performed in a unidirectional or bidirectional mode-   3. Wash nonspecific bound materials from the column using a flowing    stream.-   4. Pump an eluent through the column and through the detector to    ensure that all material has been washed from the column and a    stable detection baseline is achieved.-   5. Pump a library mixture of analytes into the column.-   6. Wash the column with an eluent to remove all excess non-attached    analytes.-   7. Optionally recover cells with analytes attached and analyze.-   8. Optionally remove analytes from column with step gradient eluent    solution. Recover, detect and analyze the analytes.-   9. Optionally remove analytes from column with continuous gradient    eluent solution. Recover, detect and analyze the analytes.-   10. Optionally recover cells from column stationary phase after    exposure to analytes and analyze.-   11. Optionally lyse cells or elute the components of the cells for    analysis. The lysis step may be done partially, or over a long time    period using gentle conditions to remove components of the cell for    processing and/or analysis.-   12. Remove and recover cells for analysis of the cells and/or    further research and development processing.

For slow kinetic interactions, the mobile phase flow rate and the linearflow velocity may be adjusted (lower) if necessary to compensate for theslower kinetic rates. These adjustments are options for all of thevarious types of chromatography.

The following steps are an example of displacement chromatographicprocedure using living cell stationary phase column.

-   1. Treat or activate column or cells to provide suitable conditions    for the column media to be able to capture cells.-   2. Load cells onto column from a flowing stream. Loading may be    performed in a unidirectional or bidirectional mode-   3. Wash nonspecific bound materials from the column using a flowing    stream.-   4. Pump an eluent through the column and through the detector to    ensure that all material has been washed from the column and a    stable detection baseline is achieved.-   5. Pump and capture analytes on the sites of the cells in the    column.-   6. Wash the column with an eluent to remove all excess non-attached    analytes.-   7. Introduce a second analyte and measure displacement of first    analyte from column.-   8. Optionally remove analytes from column with eluent solution.    Recover and analyze the analytes.-   9. Optionally recover cells from column stationary phase after    exposure to analytes and analyze.-   10. Optionally lyse cells or elute the components of the cells for    analysis. The lysis step may be done partially, or over a long time    period using gentle conditions to remove components of the cell for    processing and/or analysis.-   11. Remove and recover cells for analysis of the cells and/or    further research and development processing.

The geometry of a breakthrough curve is depicted in FIG. 9. The x-axisdenotes the time the analyte is pumped or the volume of the analytepumped. The y-axis shows the amount of analyte that exits the columnrelative the column input concentration. To start, analyte is pumpedthrough the column and is taken up by the stationary phase. The firstbreakthrough is where the analyte is not completely taken up from thecolumn. The curvature of slope A is an indication how fast the analyteis taken up by the stationary phase. A long extended slope of curve Aindicates a slow on-rate of the analyte. The slope of the breakthroughis an indication of the kinetics of uptake and release and of how thecolumn is packed. An ideal breakthrough is vertical, but in practice,all breakthrough curves have a slope. The 50% breakthrough point showshow much analyte is taken up by the column. This is calculated bymultiplying the volume at which 50% breakthrough occurs by theconcentration of the analyte being pumped through the column. Curve B isthe curve leading into the point at which no analyte is being taken upby the column, 100% breakthrough. A long extended curve at this pointindicates a slow off-rate and/or a slow on-rate of the analyte.

Competing materials can be added while performing breakthroughchromatography. Generally, small-diameter beads packed in columns withlow dead volume will give breakthrough curves with steeper slopes. Inthese cases, the mass transfer and diffusion to the surface of the beadare decreased so that the kinetic interaction and the extent ofinteraction can be measured.

The following steps are an example of breakthrough chromatographicprocedure using living cell stationary phase column.

-   1. Treat or activate column or cells to provide suitable conditions    for the column media to be able to capture cells.-   2. Load cells onto column from a flowing stream. Loading may be    performed in a unidirectional or bidirectional mode-   3. Wash nonspecific bound materials from the column using a flowing    stream.-   4. Pump an eluent through the column and through the detector to    ensure that all material has been washed from the column and a    stable detection baseline is achieved.-   5. Pump an analyte into the column. The breakthrough of the analyte    is measured by the initial breakthrough, shape and slope of the    breakthrough, 50% breakthrough point and the shape of the slope to    analyte plateau.-   6. Optionally recover cell with analyte attached and analyze.-   7. Optionally remove analyte with eluent solution. Monitor and    analyze the removal of the analyte from the cell stationary phase.-   8. Optionally recover cell after exposure to analyte and analyze.-   9. Optionally repeat with a second analyte. Pump a second analyte    through the column and measure breakthrough parameters. Measure the    difference of selectivity cell stationary phase column of first and    second analyte.-   10. Optionally, repeat with several other analytes and measure the    difference in selectivity of the different analytes.

Breakthrough curves measure effective capacity for a particular analyte,the kinetics of on interaction and the kinetics of off interaction.Analyte measurements include slope, starting and ending breakthroughshape, the breakthrough plateau shape and other parameters. Competinganalyte materials can be combined while performing breakthroughchromatography.

When the analytes are dyes that bind to specific cell surface proteins,breakthrough curves can also be used to measure the expression of cellsurface proteins. Proteins expressed on the surface of mammalian cellsare often universal. Disease cells may express cell surface proteins inconcentrations that are different from non-disease cells. In additionthe ratio of one cell surface proteins to another cell surface proteinhas been shown to be different when comparing diseased versusnon-diseased cells. This information can be used to either design newtherapeutics directed at detecting the stoichiometry of cell surfaceproteins, guide the administration of drug cocktails to individualpatients, and to diagnose disease.

In the columns and methods of the invention, cells can be captured fromrapidly flowing streams. As an example, for columns having diametersranging from 2 mm-4 mm, capture can be from cells moving through thecolumn at 0.05-20 mm/sec, 0.1-10 mm/sec, 0.2-5 mm/sec, 0.3-3 mm/sec,0.4-2 mm/sec and 0.5-1 mm/sec. The linear velocity at 0.1-10 mm/seccorresponds to absolute flow rates of about 100 μL/min to 10 mL/minrespectively. For columns with diameters ranging from 4 mm-4 mm, capturecan be performed from cells moving through the column at 0.05-20 mm/sec,0.1-10 mm/sec, 0.2-5 mm/sec, 0.3-3 mm/sec, 0.4-2 mm/sec and 0.5-1mm/sec. The linear velocity at 0.1-10 mm/sec corresponds to absoluteflow rates of about 500 μL/min to 5 mL/min respectively. For columnswith diameters ranging from 10 mm-15 mm, capture is from cells movingthrough the column at 0.05-20 mm/sec, 0.1-10 mm/sec, 0.2-5 mm/sec, 0.3-3mm/sec, 0.4-2 mm/sec and 0.5-1 mm/sec. For these columns, linearvelocity at 0.1-10 mm/sec corresponds to absolute flow rates of about 1mL/min to 100 mL/min respectively. As yet another example, for columnwith diameters ranging from 15 mm-40 mm, capture is from cells movingthrough the column at 0.05-20 mm/sec, 0.1-10 mm/sec, 0.2-5 mm/sec, 0.3-3mm/sec, 0.4-2 mm/sec and 0.5-1 mm/sec. For these columns, linearvelocity at 0.1-10 mm/sec corresponds to absolute flow rates of 5 mL/minto 500 mL/min respectively.

The following steps are an example of a chromatographic procedure forpurification of cells for recovery and detection.

-   1. Treat or activate column or cells to make the column suitable to    be able to capture cells.-   2. Load cells onto column from a flowing stream.-   3. Wash non-specific bound materials from the column using a flowing    stream.-   4. Optionally tag cells with a reagent that reacts with a group on    the surface of the cell. The tag reaches with an attachment group or    entity that is optional different than the attachment group used to    capture the cell to the column.-   5. Optionally measure cells with the cells attached to the column.    Optional detectors are infrared, surface and transmission VIS/UV,    fluorescence, chemiluminescence spectrophotometers.-   6. Optionally lyse cells or elute the components of the cells for    analysis. The lysing may be done partially, or over a long time    period using gentle conditions to remove components of the cell for    processing and/or analysis.-   7. Remove and recover cells for analysis of the cells and/or further    R&D processing, growing or transforming of the cells or further    research and development and/or therapeutic use of the cells.

Cell surface markers are used in a number of ways in columns and methodsof the invention as shown in FIG. 10. In some embodiments, cell markersare used to capture the cell to the bead for capture or to form a cellstationary phase. Other markers may be used to tagging the cells. Thiscan be performed on the column or post column recover. Other markers arethe basis for interaction of analytes with the cell stationary phase. Insome embodiments, different markers are used for different purposes asshown in FIG. 10. For example, a different marker may be used for oncolumn tagging the cell than the marker used for attaching the cell tothe bead. In this way, the so that the cell. In this way, the taggingprocess is less likely to interfere with the attachment of the cell.Similar strategies are used for cell stationary phase experiments.

Columns, instruments and methods of the invention to purify andmanipulate cells can be in different forms and configurations. Anexample of a general configuration is an instrument that operatesprimarily with back and forth fluid flow. This is shown by FIG. 11. Theinstrument may be used for cell purification, cell diagnostics or cellstationary phase chromatography.

In the figure a pipette is fitted to a column of the invention. In otherexamples, a syringe pump, gas pressurized/vacuum chamber pump orperistaltic pump can be fitted to a column of the invention. The pipettemay be manual or electronic. The electronic pipette may be controlledwith firmware and software contained within the pipette or may becontrolled by an external computer or control. The pipette may beoperated in a free standing mode with no hands or stand where the columnend is inserted into a sample containing well or assembly and back andforth flow is operated electronically and semi automatically. Thepipette is operates without manual intervention until placement into thenext solution where the pipette again operates without manualintervention.

The pipette may be a single channel or multichannel pipette. Thepipette, syringe pressure chamber or other pumping mechanism may be inthe form of an automated robotic liquid handler where a single channelis operated, multichannel are operated in parallel, or up to 96 channelsare operated in parallel. Columns of the invention may be operated oneat a time or in parallel operation two or more at a time up to 96 at atime and optionally more.

The flow of fluids through the column is bi-directional back and forthwith optional uni-directional flow for some methods. Semi-automated andautomated refer to control of fluid through the column and placement andmovement of the columns. Movement of the pipette or other pumping devicefrom well to well can be performed manually. Optionally, the flow can bedirected to a detector. The detector may measure a property of a well,or the detector may have flow through capability and a flowing stream isanalyzed.

Columns of the invention may also be used with a fully automated liquidhandler equipped to engage columns of the invention and process samplescontaining cells. The flow of fluids through the column isbi-directional back and forth with optional uni-directional flow forsome operations. Movement of the column from well to well is performedautomatically. Optionally, the flow coming out the end of the detectorcan be directed to a detector. The detector may measure a property of afluid with a well where the fluid has been deposited, or the detectormay have flow through capability and a flowing stream coming from thecolumn is analyzed.

A more general instrument where unidirectional flow and bidirectionalflow is used is shown in FIG. 12. The instrument may be used for cellpurification, cell diagnostics or cell stationary phase chromatography.

The instrument is fully automated with a flow path of eluent reservoirs,pump, injector, living cell stationary column with optional columntemperature control, detector and optional fraction or effluentcollector. The pump may be a piston, peristaltic, air or gas pressure orsyringe type pump. In some embodiments, there are two 3-way valveslocated at the column inlet and exit in order to optionally incorporatebi-directional flow through the column. Cells may be loaded onto thecolumn and purified in this manner. The liquid chromatography instrumentmay be used for purification and recovery of cells either with cellsample injected into the column with the injector or loaded using backand forth flow. The cells may be eluted and recovered from the columnand detected by changing the eluent pumped through the column. Theliquid chromatography system is very useful for studying thedifferential interactions of analytes with the cell-based solid phase.

The chromatographic purification procedure may include combinations ofunidirectional flow and bi-directional flow. For example, in theprocedures described above, the cells may be loaded or captured by thecolumn with back and forth flow and then the rest of the procedureperformed with unidirectional flow.

In this configuration, not only is the column compatible with the cellsso that they are not harmed, the tubing, tubing connections, valves,flow detectors and fraction collector are all cell compatible so thatthe cells are not harmed. This includes making certain the flow pathdoes not contain any sharp edges or surfaces that may puncture the cellwall. In addition the flow path does not contain chemicals such as metaloxides that may chemically harm cells or adsorb cells.

The liquid chromatograph may optionally include temperature control ofthe sample that is captured, fluid that is pumped through the columnand/or the column. Fluid may be pumped through the column to providenutrients and preserving the cells of the stationary phase column. Thefluid may contain glycerol and other material to slow metabolism topreserve the cells of the stationary phase column. The temperature maybe lowered to preserve the cells of the stationary phase column.

Detection may be continuous or fractions may be collected and analyzed.Analyte measurements include retention time, capacity factor,selectivity coefficient, breakthrough curve parameters. Examples ofdetectors include Cell flow Cytometer, U V and Fluorescent, RefractiveIndex, Chemiluminescence, Electrochemical Detectors, PCR or othernucleic acid measuring detector, and Mass Spectrometer Detector.

Applications of cell-based stationary phase liquid chromatographyinclude drug discovery, drug development (including personal drugdevelopment) and cell research. In drug discovery, the interaction of alibrary of compounds may be studied for a particular type of cell. Indrug development, the interaction of a particular drug candidate, classof drug candidates, or analogs of a drug candidate may be studied. Incell research, the interaction of a compound with a particular cell orcomponent of a cell may be measured and studied.

Measurement of Analytes and Cells in Cell Stationary Phase LiquidChromatography

Partitioning, gradient and displacement chromatography with a cellstationary phase may require the presence of competing entities tointeract with the cell stationary phase. In the case of an antibodyanalyte interacting with the marker on the cell surface of a stationaryphase, the eluent may contain another antibody or fragment of anantibody or a protein that has an affinity for a cell marker.

Displacement chromatography and frontal/breakthrough chromatography maybe performed with or without a competing entity. Frontal chromatographyis usually performed with unidirectional flow. These types ofchromatography may be performed at different mobile phase ionicstrengths, with different buffers and at a different pH to determine theextent of interaction of the analyte with the stationary phase.

Analytes may be small molecules or metabolic materials. These analytematerials may have rapid and reversible interactions with the stationaryphase. In these cases, partitioning, gradient, step gradient,displacement or frontal chromatography may be performed. In other cases,the interactions of the small molecule or metabolic materials may beslow and even difficult to reverse. Competitive mobile eluents, havingdifferent mobile phase ionic strengths, with different buffers may beused to determine the extent of interaction of an analyte with thecell-based stationary phase.

It is possible to study how analyte libraries interact with cells fordrug discovery, drug treatment or drug development.

Both the mobile phase analytes and the stationary phase functionalgroup(s) can be analyzed and measured in this type of chromatography. Nopreviously-described chromatography systems have this capability. Usingthe system, the interaction between analyte materials introduced intothe cell-based stationary phase and the cells themselves can becharacterized. For both the analyte and the cells, it is possible todetermine whether or not there was an interaction and the extent towhich the materials interacted. This examination can determine whichmaterial or materials interacted with the stationary phase, the mannerin which they interacted, the extent of interaction (ornon-interaction), which part of the cell stationary cell the material(s)interacted with and/or the effect of the interaction. This informationcan be exploited for a variety of downstream applications.

Stationary phase cells can be examined after they are used as astationary phase. This examination is another means to determine theinteraction of materials with a cell.

For example, a cancer cell stationary phase can be used to measureantibody interaction. The antibody may contain another material such asa toxin. The toxin can be delivered to the cell surface or into thecell.

Another example is a stationary phase comprised of bacterial cells usedto examine antibacterial agent interaction and outcome.

Another example would be a stationary phase of epithelial cells in whichcell signaling materials are used to measure interaction. After exposureto an analyte, the cell could be removed from the column and analyzed.

The interaction of an analyte with a cell may be measured by any type ofchromatography. The ability to take up an analyte can be measured. Then,after optional removal of the analyte, the cells that make up thestationary phase may be examined to study the interaction.

After chromatography is performed, cells can be removed from thestationary phase and examined to see if they remain alive or are dead.In some embodiments, cell viability can be determined on column. Stainscan be introduced to determine cell viability. The dead cells can bemeasured individually or together to determine if a reagent remainsbound to the cells. The relative number or percentage of dead cells andlive cells may be measured. The extent of damage to the cell membrane ofa dead cell may be measured optically. A dye can be added that will onlyenter the cell if it is living. The ratio of dead cells and live cellsmay be measured as a function of time after exposure to a material.

Components, such as DNA, RNA, organelles, proteins, lipids,carbohydrates, etc. of the dead cells may be examined to determine thecause of the cells changing characteristics or dying. For example, thecell may be examined to determine if a reagent or portion of the reagentis associated with the cells and if the cells are alive or dead.

The treatment and manipulation of the cells must be gentle, bothphysical treatment and chemical treatment. Normal physical and chemicalmanipulation of a cell can cause damage to a cell and cause the cell todie. Measurements of cells loaded onto a resin to make a stationaryphase but not used as a stationary phase is considered to be abackground killing of cells which can serve as a control. These cellsare not measured or their measurement is subtracted from the measurementsignal.

These steps can be used for making and using a cell-based stationaryphase.

-   1. Load or pack column with base resin beads.-   2. Load column with an antibody or cell binding reagent.-   3. Load column with cells. (In some embodiments, attach the cells to    the beads and then pack the beads into the column.)-   4. Expose column to a material with injection, block treatment, or    breakthrough treatment and optionally measure interaction of the    material with the cell stationary phase.-   5. Optionally, wash the column.-   6. Optionally, remove the cells from the column.-   7. Analyze the cells for specific properties or characteristics.    Cell Stationary Phases with Controlled Mobile Phase Concentration:

Impervious resin may also be used for cell purification. Sinceantibodies and other affinity reagents that are used to functionalizethe resin do not enter the matrix of the bead but are only in theinterstitial space and channels of the column, then the concentration ofthe reagents are effectively much higher when they enter and travelthrough the column. They are not diluted by that volume of liquid thatis within the resin matrix. Higher concentrations of the antibody,aptamer and other reagents is useful because less reagent will be neededto functionalize the resin and the conditions needed to functionalizethe resin will be lower. A higher concentration of reagents can make itpossible to drive equilibrium reactions to completion.

Cell stationary phase columns can be based on substrates that areswollen with water. These include agarose, Sepharose, dextran, celluloseand other hydrophilic polymers that swell with water. When reagents areintroduced or pumped through columns containing these substrates, cellsif present will travel to the surface. Cells may not be able to enterthe stationary phase matrix because the pores may not be large enough.However water molecules, buffer molecules and other small size reagentsmay enter the pores of the resin substrate. Reagents such as aptamers,antibodies, FABs, and other affinity reagents may enter the stationaryphase matrix.

This phenomenon can make it difficult to control of the chemicalenvironment surrounding the cells. Reagents can be concentrated if theydo not enter the pores of the substrate while reagents that enter thepore may become diluted. In any case, the concentration of anyparticular reagent after it enters the column and as it travels down thecolumn is unknown and unpredictable. But it is important to determinethe concentration of the reagents in order to practice chromatography inthe most predictable manner.

In some embodiments of the invention, the substrate used to contain thecell stationary phase is solid and impervious. Water and buffers do notenter the stationary phase matrix. Examples of this are impervioussilica and zirconia and other inorganic materials, solid imperviouspolystyrene and other polymers. With these materials, the reagents andcells do not enter the matrix or interior of the bead when the bead isexposed to reagents and cells. But rather they reside in the spacebetween the beads or in the interstitial space of the media and channelsor they reside on the surface of the media. The concentration of thereagents only changes if they react with the affinity phase and are notdiluted or concentrated by water or reagents entering the stationaryphase matrix.

In certain embodiments, an impervious resin is utilized. The reductionin non-useable surface area will decrease reagent costs and the rigidstructure will facilitate an easier packing procedure. This is shown inExample 18 which describes the synthesis of E. coli with water-swollenagarose stationary phase and an impervious silica cell stationary phase.Impervious resins can be used for the capture and recovery of cells aswell as for cell-based stationary phase liquid chromatography.

Drug Development

The cell-based stationary phase can be used for drug developmentapplications. In some embodiments, cells can be immobilized on a columnand then challenged with different entities such as libraries or poolsof molecules (e.g., small molecule drug leads or biologics). In otherembodiments, cells can be immobilized on a column and the interactionwith other cells can be examined. Conversely, drug candidates can beimmobilized on the column and challenged with different cell types.

In one example, a library of small molecule drug candidates labeled foridentification can be exposed to cells immobilized on a column. A washstep can be performed and the cells can be eluted from the column. Thosecells that have a drug candidate bound can be identified. Massspectrometry can be used to identify the drug candidate and its targeton the cell.

A number of techniques can be used to screen for potential drug leads.As mentioned previously, target cells can be immobilized on a column.When done in multiplex, multiple cell-immobilized columns can bescreened in parallel. Multiplex operation can be performed with between2 and 1536 columns simultaneously. Each column can be subjected to adifferent drug lead to screen for the desired cell interaction orsignaling event. The following techniques can be used.

-   -   1. Eluting the cells and performing qPCR (e.g., to measure a        change in gene expression or particular mRNA). For example,        expression of genes involved in programmed cell death could be        measured.    -   2. Use cells transfected with a reporter gene such as GFP or        luciferase. The reporter gene would be engineered with a        promoter region corresponding to the desired cellular event. If        the promoter is induced by a drug lead, the cell would express        the reporter. Detection of the reporter gene expression can be        performed on column or after cells are eluted from the column.        As an example, differentiation of stem cells could be measure        with a reporter gene.    -   3. If the drug is designed to induce a regenerative cellular        event, cell growth and doubling can be monitored as the final        assay. Cells could be eluted and grown in cell culture.        -   In one example, cells having a known drug target could be            used to identify potential drug as follows.        -   Put cells having a validated target on the column.        -   Challenge the cells on the column with library of            fluorescently-labeled drug candidates.        -   Wash        -   Elute cells        -   Count/separate the labeled cells in a cell sorter.        -   Analyze cells or components of cells by mass spectrometry by            LC/MS, MALDI, etc.            As an alternative to fluorescent labels, drug candidates            could be labeled with DNA barcodes. PCR could then be used            to identify particular candidates able to bind cells.

In another example, an unlabeled library can be used and drug candidatescan be identified using a cell viability assay.

-   -   Put cells having a validated target on the column    -   Challenge with library of drug candidates    -   Wash    -   Add live/dead cell stain on column    -   Wash    -   Elute cells        Alternatively, the cells can be stained after elution. The        results could be evaluated using fluorescence microscopy or flow        cytometry. A column in which the cells were not challenged with        the drug candidate could serve as a negative control.

A variety of detection methods can be applied to the methods describedherein. Non-limiting examples follow.

-   -   cell sorter or flow cytometry    -   fluorescence microscopy    -   light microscopy (e.g., to examine cell capture, viability or        morphology)    -   mass spectrometry    -   PCR    -   sequencing    -   On-column or in-line detection    -   electrophoresis

In another example it may be desirable to investigate the response ofdifferent cell types to a drug. For example, a known cancer drugeffective on one cell type may also bind to or be effective againstanother cell type. The following experiment could be performed.

-   -   Attach labelled drug to the column (or could attach cells)    -   Add cancer cells of a different type    -   Wash    -   Elute    -   Count/separate labelled cells in cell sorter

As mentioned above, partitioning may be useful in some instances. Forexample, partitioning can be used to distinguish between severalpromising drug candidates, all of which bind the cells with relativelylow affinity.

-   -   Immobilize cells on a column    -   Add mixture of several labelled drug candidates    -   Collect fractions or use in-line detection

In this experiment, the relative binding efficacy of each drug candidateis determined by its elution order. This technique can also be used tocharacterize the relative binding efficacy of different monoclonalantibodies. Cells can be immobilized on the column and challenged withdifferent monoclonal antibodies. In some embodiments, a known drug mightbe tweaked for instance by mutagenesis or synthesis. The relativebinding of different drug analogues could then be investigated usingpartitioning.

In another embodiment, the following experiment could be performed tosimultaneously identify drug targets and drug leads.

-   -   1. Immobilize cell of choice on column    -   2. Screen DNA bar-coded small molecule drug libraries,        antibodies or antibody derivatives    -   3. Present drug libraries to cell-immobilized columns    -   4. For a negative control, do not present drug libraries    -   5. Wash    -   6. Release negative control cells and cells in complex with drug        leads    -   7. Disrupt negative control cells and cells in complex with drug        leads to generate membrane fragments consisting of cell-surface        proteins and cell surface proteins bound to drug leads    -   8. Run non-denaturing gels of negative control membrane        fractions and experimental membrane fractions.    -   9. Identify, through gel shift, membrane protein-drug complexes.    -   10. Extract leads and use mass spec to identify membrane protein        and drug lead.

In certain embodiments, cells can be immobilized on the column anddisplacement chromatography can be used. For example, it may bedesirable to compete off a naturally-occurring ligand with a drug for apathway blocking drug application.

Breakthrough or frontal chromatography can be used in some instances,particularly for drug maturation studies. Breakthrough curves such asthe one shown in FIG. 9 can aid in identifying entities having thedesired binding kinetics, regardless of whether they're fast or slow.Several drug candidates or analogues can be compared in this manner.

The use of columns is advantageous for sequential additions of differentmolecules or compounds. For example, to examine calcium-dependentinteractions, calcium could be added to the cells immobilized on acolumn, followed by the addition of a library.

Liquid-Sealed Chromatographic System

The sealed chromatographic system is a liquid chromatography column thatoperates without exposure to ambient conditions. Once sealed, thecomponents of the chromatographic device and liquids within the devicecannot be contaminated by materials outside the sealed system. Ambientconditions are defined herein as the conditions of the surroundingenvironment. The sample, wash and elution solutions are passed throughthe column in a closed environment. The column can be sterile and can beused to isolate cells or enrich cells in a sterile environment. Thecolumn itself and the solutions passed through the column can besterile. The entire chromatographic process is performed under sealed orclosed conditions including sample loading onto the column, columnwashing, and column elution. The purified product is recovered in aclosed receiving container. The sealed format prevents contaminants fromentering the system. The system may contain one or more vents. Howeverthe vents must operate in a way that materials may only leave the systemand not enter the system. Examples of vents may include check valvesthat only let material out, or a 0.2 micron filter that lets gases leaveor enter but does not let bacteria or other contaminants enter thesystem.

As with other embodiments described herein, cells can be captured usingaffinity, hydrophobic interaction, reverse phase, normal phase, ionpairing, ion exchange or other strategies. Alternatively, an enrichmentcan be performed in which the desired cell types pass through the columnwhile other non-desired materials are retained.

Liquids flow from a feed bag or reservoir to a receiving bag/reservoir.Flow through the column can be bidirectional or unidirectional.Bidirectional or back and forth flow can optionally be used for any orall of the equilibration, capture, wash and elute processes. The sealedsystem does not contain flow restrictions or backpressure to flow thatwill restrict the flow of liquid with the pumps used by the system. Ofcourse, restrictions that provide backpressure that stop or slow theflow are harmful. But even small restrictions to flow are harmfulbecause they could make the flow through the system unpredictable andunreliable. A pump may appear to work with a sealed system, but changinga parameter such as the tubing, column or fitting may marginalize thesystem to render the sealed system unreliable. Sealing the inletreservoir may allow the pump to work for a time period, but may stopworking if a negative pressure develops preventing feed of the liquidinto the pump. Likewise, sealing the outlet reservoir may allow the pumpto work for a time period, but may stop working if a positive pressureincreases the resistance of flow out of the system.

FIG. 14 shows an example of a sealed column system for cell capture witha back and forth flowing system. The flow is controlled by the relativedifferences in the height of the feed and receiving closed containers.The system contains column 102 and reservoirs, 202 and 302. Depending onthe direction of flow, a given reservoir can either be a feed to column102 or the reservoir can receive flow from column 102. In someembodiments, the sealed column system may also contain on/off valves 402and 502 to allow flow or stop flow. Reservoirs 202 and 302 are connectedto valves 402 and 502 and column 102 with flexible tubing 602.

The flow through the column, 102 can be controlled by several differentoptions. In one method, the relative difference in height of feed andreceiving sealed containers will apply a positive pressure on one sideof the column and a negative pressure on the other side of the column.In FIG. 14A, reservoir 202 is positioned above column 102 and reservoir302 is positioned lower than reservoir 202. This will cause flow fromreservoir 202 through the column 102 and into reservoir 302 as depictedby the arrow below column 102. The flow rate can be increased bychanging the relative position of the two reservoirs, for example byincreasing the height of reservoir 202 or lowering reservoir 302.Reservoir 302 may be placed below column 102 or above the height ofcolumn 102.

Reversing the positions of the two reservoirs as shown in FIG. 14B willreverse the flow through column 102 as shown by the arrow going fromright to left below the column. Flow will stop when the feed reservoiris depleted or the receiving reservoir is full.

In some embodiments, flow through sealed column 102 may be powered byperistaltic pumps. For example, valve 402 and/or 502 may be replacedwith a peristaltic pump or pumps. When a peristaltic pump is used, thetubing can remain sealed. Flow through the sealed column may also beperformed with syringe pumps. For example, reservoirs 202 and/or 302 canbe replaced with syringe pumps. In this embodiment, feed and receivingchambers 202 and 302 remain sealed.

FIG. 15 shows an alternate embodiment of a sealed column system forcapture of cells with a back and forth flowing system. The systemcontains column 25 and reservoirs 5 and 15. Depending on the directionof flow, a given reservoir can either provide feed to column 25 orreceive effluent flow from the column. The reservoirs are connected tothe column through tubing which can be flexible sealed tubing. The flowthrough the column 25 is controlled by placing the reservoirs and columnon platform 55 where fulcrum 35 is positioned at or near column 25. Thereservoirs are raised and lowered relative to each other by tiltingplatform 55 at fulcrum 35.

The relative difference in height of the feed and receiving sealedcontainers will apply a positive pressure on one side of the column andnegative pressure on the other side of the column. In some embodimentsthe positive pressure of the feed reservoir can be in the range of 5psi, 4.5 psi, 4 psi, 3.5 psi, 3 psi, 2.5 psi, 2 psi, 1.5 psi, 1 psi, 0.5psi, 0.25 psi, 0.1 psi, 0.01 psi or 0.001 psi. Likewise, the pressure inthe receiving reservoir can be in the same range, however it is alsopossible for the pressure of in the receiving reservoir to be 0. Flowwill stop when the feed reservoir is depleted or the receiving reservoiris full.

FIG. 16 depicts a sealed column system for capture of cells with a backand forth flowing system with two feed and receiving containers on eachside of the column. The on/off valves control the flow into and out of aparticular container. In FIGS. 16A and 16B, valves 50 and 70 are closedand flow is between closed containers 15 and 35. FIGS. 17A and 17Bdepict the same system architecture in an alternate configuration inwhich valves 50 and 70 are open while valves 40 and 60 are closed.

FIG. 18 depicts an alternative configuration of a sealed liquidchromatography column system for capturing cells using a flowing systemwith five each feed and receiving containers. The system may be used inunidirectional flow or back and forth flow. The pumping system may begravity, pressure, vacuum, or peristaltic pumping. In thisconfiguration, there are two columns 10 and 20. Column 20 has five feedand receiving containers on each side of the column. The on/off valvescontrol the feed and receiving container that is in use and theparticular column that is used as described in FIG. 15. A specifiedpurification, washing and recovery method uses a controlled sequence ofvalves (pictured as small ovals) opening and closing. Second column 10gives the option of using a second chemical treatment on purified andrecovered cells from column 20. The sealed chromatography system isdescribed in more detail below in Example 22.

In some embodiments, a manifold can be used to introduce other liquidinto the column in a sealed system including wash and elution solutions.A third bag can be configured to receive purified cells. A second columncan be configured to clean the purified cells for materials includingantibodies, biotin, etc. with the cells received into a fourth bag. Insome embodiments, the system can be used to remove cancer cells orpathogens from blood. In all cases, back and forth flow can be used ifnecessary to achieve a complete reaction processes.

Pressures applied to the sealed chromatography system column andreservoirs are complex. The separation column has an additionalbackpressure or resistance to flow force exerted upon it. This is due tothe eluent flowing out of the column which is in direct fluid contactwith the reservoir of fluid collected from the column. The backpressurewill change depending on the relative volumes of liquid above and belowthe columns (inlet and outlets) and the positions of the inlet andoutlet volumes. It is not possible to predict the pressures as they arevariable. It is surprising that that flow of fluids can be initiated (ineither direction), maintained and established with the columnconfiguration and design constraints. It is also surprising that thecapture, washing and recovery of cells can be accomplished with variableflow rates and variable pressures. It is also surprising that theprocess can be performed reproducibly and predictably (predictablepurity, concentration and volume).

In the sealed system, resistance of liquid and cell flow through thecolumn is caused not only by the column but also by the receivingcontainer which is in intimate contact with the fluid. A positivepressure is needed to expand the receiving container as it is filledfrom the column effluent. There may also be a pressure pushing backagainst or resisting the flow caused by the receiving containerattempting to contract rather than expand. This resistance to flowthrough the column is in addition of the liquid attempting to flowthrough the column and the cells themselves having a resistance oftransversing the column. Thus, the positive force needed to pump thecells and liquid through the column must overcome all of these forces ina sealed system. The positive force pushing the liquid through thecolumn will be variable and, if gravity is used, will become exceedinglysmall, as the amount of liquid on top of the column approaches zeroforce with depletion of the liquid above the column. Nevertheless, allof the liquid can be pumped through the column.

The pumping force for a sealed system can be any peristaltic pump,positive pressure pump, piston pump, diaphragm pump, negative pressurepump, syringe pump, pipette pump, or gravity pump. The pump may forcethe liquids to flow through the system in back and forth flowbidirectional modes, unidirectional flow modes, single passunidirectional mode, circulating pass mode or combinations of these flowmodes.

Non-limiting examples of the ways in which the columns and methods ofthe invention can be used include the following.

-   1. capture and release of cells-   2. depletion of cells from a complex mixture and retention of    remaining cells-   3. capture labeled cells, then release and count-   4. Capture cells, lyse cells on column or after the cells have been    eluted from the column. Collect DNA, RNA or proteins or other    cellular components and analyze.-   5. Purify sperm from other cells or from the environment.-   6. Purify sperm from a crime scene away from other cells and other    materials and perform DNA analysis to identify the source of the    sperm-   7. After cell capture, the column is washed and cells can optionally    be reacted with a dye to label the captured cells. The resin may be    removed from the column and plated or spread on a surface. The resin    beads containing attached cells may be sorted and counted or    analyzed by any means.-   8. Capture a group or class of cells based on a specific parameter    and then perform a secondary capture step to produce and recover a    subset of cells.-   9. Capture and recover cells from blood, fluid, marrow, skin and    tissue.-   10. Capture and recovery of live cells from the examples listed    above from various samples including, blood, fluid, marrow, skin and    tissue.-   11. Capture and recovery of cells including T cells, organ cells,    and stem cells. Cells are then used for therapeutic purpose.-   12. Capture and recovery of large number of cells on a pipette tip    column having a body size in the range of 2 mL-100 mL.-   13. Therapeutic screening-   14. Cell clean-up-   15. Diagnostics for food, medical, environmental, forensics, etc.-   16. Drug discovery-   17. Drug development-   18. Cell metabolic research-   19. Organ cell device and methods where cells of a particular cell    organ type are loaded onto a column.

EXAMPLES Example 1. Sperm is Captured, Separated from Cells and DNAAnalysis is Performed

In forensics, it is often desired to obtain DNA profiles from oldstains, body fluid samples and other possible samples. The primary goalis to preferentially separate sperm from vaginal cells and othermaterials, a necessity for DNA analysis in rape cases, for example.

DNA aptamers which are short strands of DNA were developed by SomaLogic(Boulder, Colo.) to bind sperm heads, and used to both identify andimmobilize the sperm heads for purification and later DNA analysis.These aptamers are used in a column bed system of the invention withbiotin and Streptavidin linkers to selectively capture sperm cells. Theaptamer sequences bind preferentially to both the outer protein membraneand the stripped perinuclear calyx of sperm cells in the presence ofnon-sperm epithelial cells.

Sperm cells (research vials, prepared by density gradient centrifugationand subsequent washing) are purchased from California Cryobank. Washedsperm cells are prepared using three washes and suspension in a buffersupplemented with Triton X100 detergent and NaCl to final concentrationsof 1% v/v and 600 mM HeLa cells to simulate non-sperm epithelial cellsare added and the mixtures are incubated for ten minutes.

Cotton swabs are used to simulate capturing the sperm sample. The sampleis removed from the cotton swab with a buffer. Aptamers with biotinlinkers are added to the solution and incubated. After washing of thesample the mixture is passed through a streptavidin packed bed column ofthe invention. The sperm is captured and subsequently washed by passingwash buffer through the column.

The sperm is eluted from the column by passing a buffer through thecolumn breaking up the aptamer/sperm column. Eluted aptamer DNA arepurified and then amplified for DNA analysis.

Example 2. Antibody Purification of Sperm

This example uses antibodies rather than aptamers to capture sperm cellsin the presence of other cells. A cocktail of antibodies specific tosperm cell surface antigens are anchored to Protein A affinity beadspacked into a column of the invention. The specificity ofantibody-antigen binding selectively captures sperm cells from samplesthat are comprised of a mixture of sperm cells, white blood cells,epithelial cells, cell lysates, etc. Alternatively, the antibodies areadded to the sample mixture first and then captured by the column. Afterwashing with a neutral buffer, the sperm cells are eluted with low pH orhigh pH buffers and the DNA is analyzed.

The antibodies may be tagged with His tags for example. In this case,IMAC beads may be packed into columns of the invention to capture theantibodies which are used in turn, to capture the sperm. In this case,the antibody sperm combination may be eluted, the cell lysed and the DNAanalyzed. Other tags may be used such as FLAG-ANTI FLAG, etc.

Peptide tags are used for capture. These include AviTag, a peptideallowing biotinylation by the enzyme BirA so the protein can be isolatedby streptavidin, Calmodulin-tag, a peptide bound by the proteincalmodulin, FLAG-tag, a peptide recognized by an antibody, HA-tag, apeptide recognized by an antibody, Myc-tag, a short peptide recognizedby an antibody, SBP-tag, a peptide which binds to streptavidin, Softag1, for mammalian expression, Softag 3, for prokaryotic expression, V5tag, a peptide recognized by an antibody, and Xpress tag.

Covalent tags include Isopeptag which binds covalently to pilin-Cprotein and SpyTag which binds covalently to SpyCatcher protein.

Protein tags include BCCP (biotin Carboxyl Carrier Protein), a proteindomain recognized by streptavidin, glutathione-S-transferase-tag, aprotein which binds to immobilized glutathione, green fluorescentprotein-tag, a protein which is spontaneously fluorescent and can bebound by nanobodies, maltose binding protein-tag, a protein which bindsto amylose agarose, Nus-tag, Strep-tag, a peptide which binds tostreptavidin or the modified streptavidin called Strep-Tactin andThioredoxin-tag.

Example 3. Circulating Tumor Cells

A cancerous tumor sheds small numbers of tumorous cells into itsimmediate vasculature. These cells then make their way into thecirculatory system, and are thus called circulating tumor cells (CTCs).CTC information is used cancer prognosis, therapy monitoring andmetastasis research.

Circulating tumor cells (CTCs) are important targets for study tounderstand, diagnose, and treat cancers. However, CTCs are found inblood at extremely low concentrations which makes isolation, enrichmentand characterization challenging. A typical concentration in a humancancer patient is approximately 1-100 CTCs per mL of blood.

CTC purification with the columns of the invention capture many or mostof the CTCs in the blood sample (high capture efficiency) and areselective with very few other cells accidently isolated. The samples areprocessed with sufficient speed and without battering of the cells sothat cells remain viable in many cases.

The columns of the invention operate by coating the column media with anantibody (anti-EpCAM) and then bonding the antibody to the epithelialadhesion molecules (EpCAM) of CTCs. After capture of anti-EpCAM labeledCTCs from a blood sample, CTC identification and enumeration areachieved using immunostaining.

During one experiment 2 to 80 spiked breast cancer cells are isolatedfrom 1 mL of mice blood sample with 90% capture efficiency. A 200 μL bedcolumn with a 1 mL pipette tip body is used for one experiment. Wholeblood is processed through the column with bidirectional flow for 5cycles at 200 μL/min flow rate. The column is washed with buffer andthen the cells are eluted with 500 mM citric acid.

Example 4. Capture of Cells from Blood

The purification and analysis processes used in example 3 are used forother cell types including white blood cells, stem cells, T cells, Bcells and others. In this example, the medium used can be capable ofcapturing each cell type. Alternatively, the medium may capture othersample components, thereby enriching for the desired cell type.

Example 5. Capture of Cells from Blood

The purification and analysis processes used in examples 3 and 4 areused except the pumping methods for flowing the fluids through thecolumn are changed as follows. The pumping method is bidirectional,unidirectional, gravity flow and gravity flow plus vacuum and/orpressure.

In addition, the method is performed with two different columnconfigurations. In one configuration, there is an air gap above thecolumn bed, while in the other configuration, there is no air gap abovethe column bed.

Example 6. Capture of Cells from Blood

The purification and analysis processes used in examples 3, 4 and 5 areused except the column has a bed volume of 1 mL inside a 10 mL pipettebody. The flow rates are approximately 10 times faster with this columnso samples sizes approximately 10 times greater are processed inapproximately the same time.

In this example the cells are released from the column by enzymatic andchemical cleavage of the linker. The cells are collected and counted.

Example 7. Capture of Cells from Tissue

For tissue samples composed of different types of cells, heterogeneouscell populations will be present. To obtain as much information aspossible about an individual cell type, biologists have developed waysof dissociating cells from tissues and separating the various types. Amild procedure is used to collect whole, intact cells. Homogenized cellsare kept at low temperatures to prevent autolysis and kept in anisotonic solution to prevent osmotic damage.

The first step in isolating cells of a uniform type from a tissue thatcontains a mixture of cell types is to disrupt the extracellular matrixthat holds the cells together. For example, viable dissociated cells areobtained from fetal or neonatal tissues. The tissue sample is treatedwith proteolytic enzymes (such as trypsin and collagenase) to digestproteins in the extracellular matrix and with agents (such asethylenediaminetetraacetic acid, or EDTA) that bind, or chelate, theCa²⁺ on which cell-cell adhesion depends. The tissue can then be teasedapart into single living cells by gentle agitation to make a cellsuspension.

Columns of the inventions are loaded with antibodies that have anaffinity for fetal cells. The suspension is passed with bidirectionalflow through the column to capture the cells. After washing, the cellsare released with by treatment with trypsin to digest the antibodies.The cells may be visually tagged by using an antibody coupled to afluorescent dye to label specific cells.

Given appropriate surroundings, most plant and animal cells can live,multiply, and even express differentiated properties in a tissue-culturedish. The cells can be watched continuously under the microscope oranalyzed biochemically, and the effects of adding or removing specificmolecules, such as hormones or growth factors, can be explored. Inaddition, by mixing two cell types, the interactions between one celltype and another can be studied. Experiments performed on cultured cellsare sometimes said to be carried out in vitro (literally, “in glass”) tocontrast them with experiments using intact organisms, which are said tobe carried out in vivo (literally, “in the living organism”). Theseterms can be confusing, however, because they are often used in a verydifferent sense by biochemists. In the biochemistry lab, in vitro refersto reactions carried out in a test tube in the absence of living cells,whereas in vivo refers to any reaction taking place inside a living cell(even cells that are growing in culture).

Example 8. Capture of Bacterial Cells

An E. coli culture is grown at 37° C. The E. coli strain is engineeredusing recombinant DNA techniques so that surface proteins on the cellcontain histidine tags. A spike of Salmonella is added to the sample sothat the sample contains 10% Salmonella cells, 90% E. coli cells, mediaand other materials.

A 1 mL bed size column containing Ni form IMAC affinity media is used totreat or process a 3 mL sample with unidirectional single pass flowunder gravity. Some air pressure is used to push the last remainingsolution through the column. The E. coli cells are removed from themixture and are captured on the column while the Salmonella cells remainin the sample.

Example 9. Capture of Cells from Culture

Most plant and animal cells can live, multiply, and even expressdifferentiated properties in a tissue-culture dish. The cells can bewatched continuously under the microscope or analyzed biochemically, andthe effects of adding or removing specific molecules, such as hormonesor growth factors can be explored. In addition, by mixing two celltypes, the interactions between one cell type and another can bestudied. After growing the cells, the specific cells are capturedaccording to processes similar to those described in examples 7 and 8.

After capture, the column is washed and optionally reacted with a dye tolabel the captured cells. The resin may be removed from the column andplated or spread on a surface. The resin beads containing attached cellsmay be sorted and counted or analyzed by any means.

Example 10. Companion Diagnostic to Antibody or Fab Based Drug

Often it is unknown whether a particular antibody or Fab drug will beeffective against a particular cancer case. The treatment process can betrial and error, trying one drug and then if it is not effective, tryingthe next drug and so on. Columns of the invention may be used todetermine the potential effectiveness of a series of drugs. Tagged drugantibodies and Fabs are prepared. A series of columns of the inventionare prepared each with a single antibody bound through the tag to themedia of the column. In this way, each available drug is represented bya column. Then a blood sample from a cancer patient is treated by theseries of columns in an attempt to capture circulating tumor cells. Thecolumns are washed and the cells, if present, are recovered and analyzedby fluorescence, DNA, microscopy or any suitable analytical technology.Specific drugs that may be effective against the cancer are capturedcontaining drug based affinity media. Then a treatment program isdesigned using the antibody/Fab drugs that have the highest affinity forthe tumor.

Example 11. Nickel-IMAC Column Sterilization

1-ml pipette tip columns containing 80 μL of Ni-IMAC resin weremanufactured and 100% glycerol was added to the columns so that theglycerol filled the resin bed up to several millimeters above the bed.The columns were placed in a pipette tip box and the box was autoclavedat 132° C. for 45 minutes.

To verify column sterility, the column resin and a piece of the columnbody were placed on an LB plate and the plate was incubated at 37° C.After 3 days, no bacterial growth was evident.

The performance of the autoclaved columns was compared with identicalcolumns that had not been autoclaved. His-tagged enhanced greenfluorescent protein (eGFP) was purified on the columns using an E4 XLSpipette (Rainin) as shown in Table 1.

After the sample was loaded onto the columns, two washes were performed.The column was washed with 10 mM NaH₂PO₄, 5 mM Imidazole, 0.3 M NaCl, pH7.4 followed by 10 mM NaH₂PO₄, 0.3 M NaCl, 20 mM Imidazole, pH 7.4. Nextthe eGFP was eluted with 10 mM NaH₂PO₄, 0.14M NaCl, 0.25M Imidazole, pH7.4.

TABLE 1 E4 XLS pipette settings: Volume mL Cycles Speed Equilibration0.50 1 Medium Sample 0.50 4 Medium Wash 1 0.50 2 Medium Wash 2 0.50 2Medium Elute 0.24 4 Medium

The absorbance of the eluate was read at 488 nm and the yield ofpurified eGFP was determined. Both the autoclaved column and the columnthat had not been autoclaved were able to capture eGFP as shown in Table2 below.

TABLE 2 Columns 1 and 2 were autoclaved and columns 3 and 4 were notautoclaved. eGFP Concentration Volume Yield Column excitation peakAbsorbance mg/mL mL mg 1 488 0.075 0.331 0.733 0.242 2 488 0.074 0.3260.741 0.242 3 488 0.081 0.357 0.753 0.269 4 488 0.079 0.348 0.740 0.258

Example 12. Protein-A Column Sterilization

1-ml pipette tip columns containing 80 μL of ProA resin (PhyNexus, Inc.,San Jose, Calif.) were placed in a pipette tip box and the box wasautoclaved at 132° C. for 45 minutes. Some of the columns containedglycerol while others did not.

To verify column sterility, the column resin and a piece of the columnbody were placed on an LB plate and the plate was incubated at 37° C.After 3 days, no bacterial growth was evident.

IgG was loaded on the columns and the columns were operated with an E4XLS pipette (Rainin) as shown above in Table 1. After the sample wasloaded, the columns were washed with phosphate buffered saline (Wash 1)and 140 mM NaCl (Wash 2). The protein was eluted with

200 mM PO₄, 140 mM NaCl @ pH 2.5.

The protein yield from the autoclaved columns was compared with theyield obtained from identical columns that had not been autoclaved. Theyield of purified IgG obtained the autoclaved proA columns wascomparable to those columns that had not been autoclaved.

Example 13. Column Sterilization

Four, 1-ml pipette tip columns containing 80 μL of agarose resin wereautoclaved at 132° C. for 45 minutes. Two different agarose resins wereused; agarose 1 and agarose 2. Prior to autoclaving, glycerol was addedto one column of each resin type. After autoclaving, it was observedthat the resin dried out in the columns that did not contain glycerolwhile the two columns with glycerol added remained wet as shown in Table3.

TABLE 3 Column sterilization Resin Glycerol Autoclaved Dried OutAgarose1 No Yes Yes Agarose1 Yes Yes No Agarose2 No Yes Yes Agarose2 YesYes No

Example 14. Negative Isolation of Stem Cells

In this example, a sample is enriched for stem cells by removing othercell types. A column is assembled in which the medium is comprised ofantibodies that bind undesired cells. The sample is loaded onto thecolumn and the undesired cells are captured on the column. The columnflow-through is collected and shown to be enriched for the desiredcells.

Example 15. Negative Selection of T Cells from Peripheral BloodMononuclear Cells

T cells are isolated by depletion of all non-T cells such as B cells,macrophages, and natural killer cells. Peripheral blood mononuclearcells (PBMC) are incubated with a mixture of monoclonal antibodies tocoat unwanted cells. The suspensions are then loaded onto a columncomprised of IgG, which binds the antibody-coated cells. The columnflow-through is collected and shown to be enriched for the T cells.

Example 16. Negative Selection of T Cells

The method from Example 15 is applied to different samples such as cellsfrom spleen, lymph node, tumor, and peritoneal fluid.

Example 17. Purification of Sperm Cells by Aptamer Binding

An RNA aptamer having the following sequence is synthesized. GgcagtccgtccgtcAZCGA CGCGZGZGZG ZZZGZCZZCZ ZGZZZGZZGZ CGZGCgccag aagcagaagg acg Zis the modified base, 5-(N-benzylcarboxyamide)-2′-deoxyuridine. Aphotocleavable linker is conjugated in between the beads and the aptamerto elute the bound cells from the RNA aptamer in one step by UVirradiation.

The synthesized aptamer is incubated with streptavidin beads in a 1 mlpipette-tip column containing 80 μl of streptavidin resin. A 1 ml samplecomprised of sperm cells mixed with other cells is eluted from a swabobtained from sexual assault evidence and diluted in 40 mM Hepes pH 7.5,350 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 0.1% Triton X100 detergent.

The sample is aspirated and expelled from the pipette tip column threetimes at a rate of 150 μl/min. Non-specifically bound material isremoved from the column by washing the column three times with 1 ml of40 mM Hepes pH 7.5, 350 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 0.1% TritonX100. To cleave the aptamer and cells from the resin, the column issubjected to UV irradiation at 1050 mW/cm2 for 5 min. The cells areeluted by aspirating and expelling two times with 500 μl of 40 mM HepespH 7.5, 350 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 0.1% Triton X100detergent.

Example 18. Biotinylated Silica Resin

The procedure outlines the creation of antibody conjugated silica beadsfor capture of E. coli. Silica beads were conjugated to biotin.Subsequently, the beads were reacted with aqueous avidin and then E.coli antibody tagged with biotin.

Piranha Solution Treatment

Goal: Adding hydroxyl groups to surface of silica.

-   -   SIGMA g4649-100G 106 um silica beads acid washed    -   95% sulfuric acid stock    -   30% peroxide stock    -   Methanol

-   1. Slowly add 13.1 mL sulfuric acid to 11.9 mL water to make 50%    sulfuric acid.

-   2. Slowly add 25 mL 30% peroxide solution to 50% sulfuric acid    solution to make Piranha solution.

-   3. Add 25 grams of silica beads to solution and mix every 15 minutes    for 60 minutes at room temperature.

-   4. Add silica bead solution to Buchner funnel with #2 filter paper.

-   5. Wash with dH₂O.

-   6. Wash with methanol.

-   7. Store powder in beaker at room temperature to dry.    Silanol Conjugation

Goal: Adding amine groups to surface of silica by reacting silanol withhydroxyl groups.

-   -   Toluene    -   APES (3 aminopropyl triethoxy silane)    -   Alcohol (Anhydrous Reagent 97% Isopropanol)

-   1. Add glass beads to round bottom flask.

-   2. Mix 10 mL APES with 90 mL toluene and add it to flask.

-   3. Spin intermittently on rotovac for 2 hours at room temperature.

-   4. Add to Buchner funnel with #2 filter.

-   5. Wash with alcohol (Anhydrous Reagent 97% Isopropanol).

-   6. Store powder in beaker at room temperature to dry.    Biotin Conjugation

Goal: Adding biotin to surface of silica by reacting NHS with aminegroups.

-   -   CAYMAN CHEMICAL COMPANY Item 13315 Biotin NHS (Biotin        N-Hydroxysuccinimide Ester)    -   DMSO    -   Alcohol (Anhydrous Reagent 97% Isopropanol)

-   1. Dissolve 500 mg biotin NHS in 100 mL DMSO.

-   2. Add glass beads to 250 mL round bottom flask.

-   3. Add 50 mL biotin solution to round bottom flask.

-   4. Mix by hand every 8 minutes for 2 hours at room temperature.

-   5. Add to Buchner funnel with #2 filter

-   6. Wash with alcohol (Anhydrous Reagent 97% Isopropanol).

-   7. Store powder in 50 mL conical tube in refrigerator.

Example 19. Packing Pipette Tip Columns

Column Packing—Antibody Pipette Tip Columns

-   1. Add powdered biotin-conjugated resin to tip body.-   2. Dispense alcohol (Anhydrous Reagent 97% Isopropanol) into pipette    tip column.-   3. Store in 50 mL conical tube at room temperature    Column Packing—Ion Exchange Tip Columns Strong Base Anion Exchange    Resin Tip Columns-   1. Suspend strong anion exchange agarose resin in water.-   2. Aliquot into reservoir-   3. Mix with pipette then pipette 500 uL into Eppendorf tube.-   4. Continually add resin to column body and allow to drain until 500    uL has been added-   5. Keep in 50 mL tube with dH₂O at room temperature

Example 20. Capturing and Eluting E. coli Cells on Pipette Tip Columns

Loading E. coli Cells onto the Column Substrates

Experiment

Using pipette tip columns packed with experimental avidin resin,determine whether DH5α E. coli cells can be captured. Comparepurification process with pipette tip columns packed with ion exchangeagarose.

Prepare Sample

-   1) Inoculate 5 mL TB medium with single colony of DH5α.-   2) Incubate at 37° C. for approximately 16 hours with shaking at 350    rpm.-   3) Measure absorbance at 600 nm until reading reaches 2.4.-   4) Aliquot 1 mL culture into 1.5 mL microfuge tubes.-   5) Centrifuge at 5,000 rpm for 5 minutes or until medium is clear.-   6) Discard medium with a pipette tip.-   7) Wash cells—resuspend in 1 mL H₂O, centrifuge at 5,000 rpm for 5    minutes, and discard water.-   8) Repeat wash two more times for a total of 3 washes.-   9) Resuspend cells in 0.5 mL water.-   10) Measure absorbance at 600 nm.-   11) Make a dilution to a concentration of 1×10⁹ cells/mL or OD 1.4.    Label “Concentrated Cells”-   12) Sample=100 μL of Concentrated cells+400 μL H₂O, or 100 million    cells.    Conjugate Avidin to Biotin Pipette Tip Columns-   1) Equilibrate pipette tip columns with 500 μL phosphate buffered    saline (PBS), 1 cycle, 0.5 mL/min, 20 second pauses.-   2) Prepare avidin    -   Avidin is supplied as lyophilized 5 mg powder (Prospec,        pro-500-a)    -   The molecular weight of avidin is 68 kDa, the bottle contains        73.5 nmoles of avidin    -   Use a syringe needle to vent the bottle    -   Use another syringe and needle to resuspend the powder in 735 uL        H₂O to make a 100 uM solution. Make 100 μL aliquots and freeze.    -   Make 100 uL of 10 uM avidin: 10 μL 100 uM avidin+90 uL H₂O.    -   Load 100 picomoles per pipette tip column: 10 μL 10 μM        avidin+240 μL H₂O-   3) Load avidin onto biotin-derivatized pipette tip columns    -   Capture with 4 cycles, 0.5 mL/min, 20 second pauses-   4) Wash pipette tip columns with 500 μL PBS, 1 cycle, 0.5 mL/min, 20    second pauses.-   5) Load biotin-antibody (Pierce, PA1-73035)    -   Antibody is supplied as 1 mL solution at 4 mg/mL    -   The molecular weight of IgG is 150 kDa    -   The solution is 0.0267 μM    -   Make 200 μL aliquots and freeze.    -   Load 0.5 picomoles of Antibody per pipette tip column: 18.7 μL        of 0.0267 μM IgG+231.3 μL H₂O    -   Capture with 4 cycles, 0.5 mL/min, 20 second pauses-   6) Wash pipette tip columns with 500 μL PBS, 1 cycle, 0.5 mL/min, 20    second pauses.    Load Cells-   1) Equilibrate pipette tip columns with 500 μL PBS, 1 cycle, 0.5    mL/min, 20 second pauses.-   2) Load pipette tip columns with 500 μL Cell Solution, 4 cycles, 0.5    mL/min, 20 second pauses.-   3) Wash pipette tip columns with 500 μL PBS, 1 cycle, 0.5 mL/min, 20    second pauses.-   4) Elute    Avidin pipette tip columns: 300 μL Buffer C (200 mM phosphate pH    2.5, 140 mM NaCl), 3 cycles, 0.5 mL/min, 20 second pauses    Ion exchange pipette tip columns: 300 μL 1M Na₂SO₄, 3 cycles, 0.5    mL/min, 20 second pauses    Analyze-   1) Visual inspection    -   Note sample turbidity after each capture cycle    -   Note turbidity of elution-   2) Measure absorbance at 600 nm on disposable plastic 1.5 mL    cuvettes.    E. coli Column Capture Loading Experiment Results

The results indicated that cells can be captured and eluted with pipettetip columns. Six samples were run through the columns using the MEAworkstation (PhyNexus, Inc. San Jose, Calif.) as described above. Therewere three ion exchange columns (Q1-Q3) and three antibody-based columns(A1-A3). Each column was exposed to 100 million cells during the capturecycle and the average number of cells eluted was in the range of 50million (FIG. 13). The concentration of each individual sample wasmeasured via OD₆₀₀ reading which was then converted to concentration permL via a ratio calculated from previous experiments. E. coli is capturedon a strong anion exchange resin due to surface markers on the cell thatcontained anion exchange groups.

Six samples were run through the six columns and the number of cells inthe sample flow-through, wash, and elution were counted via OD readingfor each column. Very few cells were released in the washing stageindicating that both resins were able to retain cells until the elutionstep.

TABLE 4 The total number of cells counted is in the range of 95 million.Total Million Sample Cells Counted Q1 114 Q2 65 Q3 84 A1 67 A2 71 A3 116Original 95

The data indicate that the creation of a cell capture pipette tip columnwas successful. The cell stationary phases are now ready to be used forchromatography.

Example 21. Evaluation of Stem Cell Multi-Potency

One method to evaluate stem cell multi-potency is to measure expressioncell surface antigens e.g. CD105, a positive marker for hMSC. Thesesurface antigens can be used to purify multipotent stem cells from amixture of non-multipotent stem cells and other material. An antibody toCD105 or other specified surface markers can be attached to the affinityresin. The cell culture is passed through the column to capture themultipotent stem cells. Non-specifically bound materials, cells andreagents, are washed from the column. Finally, the purified multipotentstem cells are eluted from the column. The cells are viable and readyfor use for therapeutic or research applications.

Example 22. Step Gradient Liquid Chromatography with Pipette Tip CellStationary Phase Columns

An eight-channel E4 pipette (Mettler Toledo) is fitted to pipette tipcolumn outfitted with adapters that allows them to fit into a 96-welldeep well plate. The pipette is programmed with firmware and software tobe about to operate with back and forth flow while freestanding in thewell. The pipette is moved manually from well to well for the variousoperations. The 1000 mL pipette tip columns are packed with 100 μL bedvolume of PS/DVB resin beads with an average diameter of 50 μm. Thebeads are surface reacted with streptavidin functional groups.

Cancer cell lines are used as a model for anti-cancer drug testing. Apanel of cell lines derived from lung cancer is tested systematically.The panel consists of cells that grow in suspension such as COR-L26. Thecells are biotin tagged for immobilization to streptavidin-derivatizedbeads. Terminal amines of cell surface proteins are labeled, on averageof one label per cell, using NHS biotin tags available from ThermoFisher Pierce (Cat. #20217).

An antibody or biomolecule library is screened for binding to the cellstationary phase columns. The library is injected on the column withbidirectional flow. Step gradient chromatography is used to elute theantibody or biomolecule from the cell stationary phase with increasingstringency steps. The antibody drug leads or biomolecules are selectedthrough their stringency binding properties. Antibodies with the lowestselectivity for the column are eluted first. The strength of the eluentis increased to elute or displace tighter binding antibodies orantibodies or other molecules that have a higher selectivity for thestationary phase. The highest selectivity or the tightest bindingantibodies or biomolecule are eluted last. The relative affinity of themolecules is measured and compared.

Pipette tip columns are equilibrated in 1 mL of PBS buffer using twocycles of back and forth flow. A cycle consists of aspiration of thebuffer volume minus 50 μL at a flow rate of 250 μL per minute, pause of20 seconds, dispense of the buffer volume minus 50 μL at a flow rate of250 μL per minute, pause of 20 seconds, while maintaining the end of thepipette tip column 1 mm above the bottom on the 96-well deep-well plate.

Pipette tip columns are moved to wells containing 1 mL of biotin-labeledCOR-L26 cells. The cells are immobilized to the beads using 4 cycles ofcapture to form the cell stationary phase column. The pipette tipcolumns are moved to wells containing 1 mL of PBS buffer and the resinis washed using 1 cycle.

The pipette tip columns are next moved to wells containing a 1 mLsolution consisting of a commercially available antibody library. Thepipette tip columns are loaded with antibody using 4 cycles of capture.

Stringency elution is performed by subjecting the pipette tip columns toa step gradient chromatography, each buffer with a lower pH. The pipettetip columns perform 4 cycles in 300 μL of PBS buffer adjusted to pH 7.2,7.0, 6.8, 6.6, 6.4, 6.2, 6.0, 5.8, 5.6, 5.4, 5.2, and 5.0 in a step-wisemanner. Each condition will be analyzed by mass spectrometry to identifyantibodies released under those conditions. The eluted antibodies may beanalyzed with MALDI mass spec, or they may be digested with an enzymeand analyzed with LC-MS. In another set of experiments, the elutiongradient is a single step gradient after an optional wash step.

In another set of experiments, a living cell stationary phase column issubjected to a potential binding antibody or biomolecule under specifiedcapture conditions or a specified set of capture conditions. The aftercapture of the antibody or biomolecule, the column is washed and theantibody or biomolecule is eluted and the amount measured. The abilityto capture the antibody or biomolecule under different chemicalconditions is a measure of the affinity of the antibody or biomoleculefor the cell stationary phase.

Example 23. Step Gradient Liquid Chromatography with Flow Through CellStationary Phase Columns

Experiments described in example 22 are performed with flow through cellstationary phase columns and apparatus as described in thespecification.

Example 24. Sealed Chromatographic System

A column was assembled by gluing a frit on one end, packing the columnand then gluing the frit on the top of the column. A 37 micron pore, 60micron thick Nitex screen frits was attached to the end of an acrylictube 0.750 inches long, 0.500 inch outer diameter and 0.375 inch innerdiameter. The tube with frit side down was placed on a deep well platefor packing. Packing was accomplished by standing the column on a standwith hole beneath that allowed the flow of liquid out of the lower end.An aqueous slurry of agarose resin, 45-165 micron particle size, wasplaced into the column by pipette. The packing material was notcompressed. Excess liquid drained away filling the column with resin.Additional slurry was added until the bed of the column reached the topof the column. The end frit of a Nitex screen of the same material wasglued onto the column end using a methylene chloride solvent.

Silicone tape was wrapped round the column to increase the diameter.Then two 10-mL plastic syringe bodies and male luer connections were cutto the 1 mL volume mark and placed on the end of the column. The columnbody was wrapped with stretchable silicone tape to seal the column body.

Male luer connections were connected to the inside of clear flexibleTygon tubing 0.25 inch outer diameter with pinch valve. The tubing wasconnected to 1000 mL Kendall cat no 763656 adapted to be a flexibleclosed feed/receiving bag containers. 250 mL of DI water was added toone of the containers and sealed. At this point, no air remained in thesystem and the entire column and receiver/feed system was closed to theoutside.

The feed bag was filled with 250 mL of DI water with the tube pinchvalve pinched closed and placed on a stand. The receiving bag was placedon stand 10 inches below the feed bad. The pinch valve was opened andthe fluid flowed from the feed bag through the feed bag into thereceiving bag in a closed system. Flow was controlled by the relativedifference in height of the two bag containers. Flow as low as a coupleof μL/min was employed to as high as 20 mL/min for this particularcolumn. Higher flow rates are possible with greater height differences,lower backpressure columns or the use of peristaltic or bag compressionpumping.

After the flow through the column was completed, the relative positionof the feed bag and receiving bag was reversed by simply lowering thefeed bag (now the receiving bag) and raising the receiving bag (now thefeed bag), reversing the flow through the column. This can process canbe performed as often as necessary to provide complete capture of thecell or other process.

In another set of experiments, the flow through the column is performedwith a peristaltic pump or other type of pumping device. In thisapparatus, the relative positions of feed and receive reservoirs is notimportant.

The invention claimed is:
 1. A method of making a cell-basedchromatography column and performing chromatography, comprising thesteps of: a) providing at least one column, wherein the column iscomprised of a packed bed of particles, wherein the packed bed ofparticles has a bed volume, wherein the packed bed of particles isretained between two frits, a lower frit and an upper frit, wherein thepacked bed of particles is comprised of physical channels, and whereinthe particles are capable of capturing cells; b) providing a biologicalsample comprised of viable cells, wherein the biological sample has asample volume, wherein the sample volume is larger than the bed volume;c) aspirating the biological sample through the column usingbidirectional flow, wherein the sample is aspirated through the lowerfrit, into the packed bed of particles and then through the upper frit;d) expelling the biological sample, wherein the biological sample isexpelled through the upper frit, into the packed bed of particles andthen through the lower frit; e) repeating steps (c) and (d) multipletimes, wherein the viable cells pass through the physical channels inthe packed bed of particles without being trapped, wherein a portion ofthe viable cells are captured on the particles, whereby the viable cellscaptured on the column comprise the cell-based chromatography column; f)performing chromatography by providing a second sample, wherein thesecond sample is comprised of antibodies, wherein the antibodies arefurther comprised of a drug or drug candidate, wherein the drug or drugcandidate is specific for a cell surface marker present on diseasedcells; and g) passing the second sample through the cell-basedchromatography column, wherein the second sample is passed through thechromatograph using a chromatography method selected from the groupconsisting of step gradient chromatography, displacement chromatography,partitioning chromatography and breakthrough curve chromatography. 2.The method of claim 1, wherein step (g) is performed usingunidirectional flow.
 3. The method of claim 1, wherein step (g) isperformed using bidirectional flow.
 4. The method of claim 1, whereinfollowing step (g), the cells are removed from the column and analyzed.5. The method of claim 1, wherein the particles in the packed bed areimpervious to reagents.
 6. The method of claim 1, wherein the method isautomated.
 7. A method of making a cell-based chromatography column andperforming chromatography, comprising the steps of: a) providing atleast one column, wherein the column is comprised of a bed of particles,wherein the bed of particles is retained between two frits, a lower fritand an upper frit, wherein the bed of particles is comprised of physicalchannels, and wherein the particles are capable of capturing cells; b)providing a biological sample comprised of viable cells, wherein theviable cells are comprised of a surface marker; c) aspirating thebiological sample into the column through the lower frit; d) expellingthe biological sample out of the column through the lower frit; e)repeating steps (c) and (d) multiple times, wherein the viable cellspass through the physical channels in the bed of particles without beingtrapped, wherein a portion of the viable cells are captured on theparticles in the bed of particles, wherein the viable cells are attachedto the particles via the surface marker, whereby the viable cellscaptured on the column comprise the cell-based chromatography column; f)performing chromatography by providing a second sample, wherein thesecond sample is comprised of antibodies, wherein the antibodies arefurther comprised of a drug or drug candidate, wherein the drug or drugcandidate is specific for a cell surface marker present on diseasedcells; and g) passing the second sample through the cell-basedchromatography column, wherein the second sample is passed through thecell-based chromatography column using bidirectional flow.
 8. The methodof claim 7, wherein the particles impervious to reagents.
 9. The methodof claim 7, wherein the method is automated.
 10. The method of claim 1,wherein the viable cells are cancer cells.
 11. The method of claim 7,wherein the viable cells are cancer cells.
 12. The method of claim 1,wherein the antibody drug or antibody drug candidate is specific forinfectious disease, diabetes, heart disease, Parkinson's, Alzheimer's orliver disease.
 13. The method of claim 7, wherein the antibody drug orantibody drug candidate is specific for infectious disease, diabetes,heart disease, Parkinson's, Alzheimer's or liver disease.